Methods and products related to 16-HETE analogs

ABSTRACT

The present invention includes 16-HETE analogs which are agonists and antagonists of 16-HETE. The compositions may be formulated in pharmaceutically acceptable formulations. The invention also includes methods and products for inhibiting neutrophil adhesion and neutrophil aggregation using the 16-HETE agonists. One method of the invention involves the administration of a 16-HETE agonist in combination with a thrombolytic agent to a patient suffering from thromboembolic stroke.

This applications claims benefit of Prov. No. 60/085,602 filed May 15,1998.

FIELD OF THE INVENTION

The present invention relates to 16-HETE analogs which are agonists andantagonists of 16-HETE. The invention also relates to methods andproducts for inhibiting neutrophil adhesion and neutrophil aggregationusing the 16-HETE agonists.

BACKGROUND OF THE INVENTION

The inflammatory response is an important element of a host's naturaldefense mechanism against pathogens. It also is involved in woundhealing. Despite the beneficial role that the inflammatory responseplays in host survival, excessive inflammation may have clinicallyadverse results in some medical conditions.

Leukocytes are a major cellular component of inflammatory and immuneresponses. This class of cells includes neutrophils, lymphocytes,monocytes, eosinophils, and basophils. Neutrophils, which play a keyrole in the inflammatory response, are generally present within the bodyin a resting unstimulated state. Once stimulated, the neutrophilsmigrate to the site of injury and release toxic factors.

The migratory capability of a neutrophil is dependent on the ability ofthe neutrophil to alter its adhesive properties. In a restingunstimulated state a neutrophil is not adhesive and cannot migrate. Oncethe neutrophil has been stimulated, however, it becomes more adhesiveand is capable of migrating. The increase in neutrophil adhesivenesscauses the stimulated neutrophil to aggregate and to adhere toendothelium. Stimulation of the neutrophil also causes the neutrophil toundergo diapedesis, which involves the migration of the neutrophilbetween post-capillary endothelial cells into the tissues.

In the tissues, an activated neutrophil releases enzymes such ascollagenase and elastase, among others. Neutrophil stimulation may alsoinitiate a burst of oxygen consumption, with concomminant activation ofthe hexose-monophosphate shunt and activation of nicotinamide-adeninedinucleotide phosphate (NADPH) oxidase. Activation of these systemsresults in the formation and release of factors such as hydrogenperoxide and hydroxyl radicals, which are toxic to microorganisms andtumor cells, and thereby facilitating the destruction of the injurycausing agent.

Several studies have focused on analyzing the control and regulation ofthe adhesive properties of neutrophils. Much of this research hascentered on adhesion receptors and also on metabolites of arachidonicacid such as C20 carbon fatty acid found in every cell membrane.Arachidonic acid metabolism occurs by different mechanisms in stimulatedversus unstimulated neutrophils and results in the production of adifferent spectrum of metabolites in stimulated versus unstimulatedneutrophils.

In stimulated neutrophils, the cytochrome P450 mixed function oxidasesystem appears to be more active. Moreover, during neutrophilstimulation, 5-lipoxygenase is translocated to the membrane compartmentfraction, where it produces 5-hydroperoxyeicosatetraenoic acid(5-HPETE). 5-HPETE is then either metabolized to5-hydroxyeicosatetraenoic acid (5-HETE) by peroxidase or dehydrated toform leukotriene A₄. Leukotriene A₄ is converted into leukotriene B₄which is a potent chemotactic agent and promoter of neutrophil adhesion.

In unstimulated neutrophils, the metabolism of arachidonic acid ismarkedly different than that in stimulated neutrophils. The metabolismof arachidonic acid in unstimulated neutrophils is sensitive tocytochrome P450 inhibitors but not to cyclooxygenase or lipoxygenaseinhibitors. Hatzelmann and Ullrich characterized the metabolitesproduced in unstimulated neutrophils, reporting the finding thatarachidonic acid is metabolized to 20-HETE and 15-HETE. Hatzelmann, Eur.J. Biochem. 173, 445-452 (1988). Another study, Kraemer et al., foundthat the arachidonic acid metabolic products formed in unstimulatedneutrophils exhibited a potent anti-aggregatory activity toward humanneutrophils, suggesting that the identified arachidonic acid metabolitesmay play some role in the regulation of neutrophil adhesion andaggregation properties. Kraemer et al., Am. J. Pathol. 128, 446-454(1987).

SUMMARY OF THE INVENTION

It was recently discovered in co-pending U.S. patent application Ser.No. 08/652,327, filed May 22, 1996 and issued as U.S. Pat. No. 5,753,702on May 19, 1998 and PCT Patent Application No. PCT/US97/08865, and itsrelated national Stage U.S. patent application Ser. No. 09/194,166, theentire contents of which are hereby incorporated by reference, that16-HETE (16-hydroxyeicosatetraenoic acid) is a component of arachidonicacid metabolism in neutrophils and that 16-HETE is a potent inhibitor ofneutrophil adhesion and neutrophil aggregation. It was also disclosed inthese applications that 16-HETE when administered alone actually reducesthe size of brain infarcts resulting from acute stroke relative to thesize of brain infarcts which occur in the absence of a therapeutic. When16-HETE is administered in combination with clot lysing thrombolyticagents such as tPA, the therapeutic combination actually reduces thesize of brain infarcts resulting from acute stroke relative to the sizeof brain infarcts which occur in a subject suffering from an acutestroke who has been treated only with a thrombolytic agent such as tPA.

The present invention relates to novel analogs of 16-HETE. Some of theanalogs of 16-HETE are agonists which maintain the biological activityof 16-HETE but which are more stable and have longer half-lives. The16-HETE analogs also include 16-HETE antagonists which inhibit theactivity of 16-HETE. These antagonists are useful when it is desirableto prevent inhibition of neutrophil activity.

According to one aspect of the invention, compositions are provided.These compositions include the following 16-HETE analog:

wherein R is selected from the group consisting of —C(O)—X—SO₂—R₁,—C(O)—X—CO—R₁, —C(O)—X—C(OH)₂—R₁, —C(O)—X—C(NH)₂—R₁, —C(O)—X—C(NH₂)₂—R₁,piperonyl, —CN, —OR′, —SR′, —NO₂, —NR′R′, amino acid, —C(O)R′, —C(S)R′,—C(O)OR′, —C(S)OR′, —C(O)SR, —C(S)SR′, —C(O)N(R′)₂, —C(O)C(O)R′,—C(S)C(O)R′, —C(O)C(S)R′, —C(S)C(S)R′, —C(O)C(O)OR′, —C(S)C(O)OR′,—C(O)C(S)OR′, —C(O)C(O)SR′, —C(S)C(S)OR′, —C(S)C(O)SR′, —C(O)C(S)SR′,—C(S)C(S)SR′, —C(O)C(O)N(R′)₂, —C(S)C(O)N(R′)₂, —C(O)C(S)N(R′)₂, or—C(S)C(S)N(R′)₂; wherein X is selected from the group consisting of O,N, and a bond; wherein R₁, R₂, and R₃ each independently is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy,aryl, and heteroaryl; wherein each R′ is (CH₂)_(z)—NR″R″ and wherein R″is independently selected from the group consisting of (C₁-C₆) alkyl,(C₁-C₆) alkenyl, (C₁-C₆) alkoxy, (C₁-C₆) alkynyl, (C₆-C₂₀) aryl,(C₆-C₂₀) substituted aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆)alkaryl, and (C₅-C₇) heteroaryl.

16-HETE analogs include both agonists and antagonists. In someembodiments the 16-HETE agonists have the following general structure:

wherein either R₁ or R₂ is a C₃ alkyl and the other is a hydrogen.

In one embodiment R₁, R₂, and R₃ each independently is selected from thegroup consisting of hydrogen, (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, and (C₁-C₆) alkoxy.

In another embodiment the 16-HETE analog has the following structure:

In a preferred embodiment X is NH and R₂ and/or R₃ is hydrogen. Inanother preferred embodiment R₃ is a C₃ alkyl. Preferably, the 16-HETEanalog has the following structure:

In a preferred embodiment X is O and R₂ and/or R₃ is hydrogen. Inanother preferred embodiment R₃ is a C₃ alkyl.

In other preferred embodiments the 16-HETE analog is one of thefollowing structures:

In another embodiment the 16-HETE analog is a 16-HETE antagonist havingthe following general structure:

wherein R₄ and R₅ each independently is selected from the groupconsisting of hydrogen, C₁₋₂ alkyl, C₄₋₆ alkyl, alkenyl, alkynyl,alkoxy, aryl, and heteroaryl and wherein when either R₁ or R₂ is a C₃alkyl the other is not a hydrogen.

According to another aspect of the invention the 16-HETE analog has thefollowing structure:

wherein R is selected from the group consisting of —C(O)—X—SO₂—R₁,—C(O)—X—CO—R₁, —C(O)—X—C(OH)₂—R₁, —C(O)—X—C(NH)₂—R₁, —C(O)—X—C(NH₂)₂—R₁,piperonyl, —CN, —OR′, —SR′, —NO₂, —NR′R′, amino acid, —C(O)R′, —C(S)R′,—C(O)OR′, —C(S)OR′, —C(O)SR, —C(S)SR′, —C(O)N(R′)₂, —C(O)C(O)R′,—C(S)C(O)R′, —C(O)C(S)R′, —C(S)C(S)R′, —C(O)C(O)OR′, —C(S)C(O)OR′,—C(O)C(S)OR′, —C(O)C(O)SR′, —C(S)C(S)OR′, —C(S)C(O)SR′, —C(O)C(S)SR′,—C(S)C(S)SR′, —C(O)C(O)N(R′)₂, —C(S)C(O)N(R′)₂, —C(O)C(S)N(R′)₂, or—C(S)C(S)N(R′)₂; wherein X is selected from the group consisting of O,NH, and a bond; wherein R₁, R₂, and R₃ each independently is selectedfrom the group consisting ol hydrogen, alkyl, alkenyl, alkynyl, alkoxy,aryl, and heteroaryl; wherein each R′ is (CH₂)_(z)—NR″R″ and wherein R″is independently selected from the group consisting of (C₁-C₆) alkyl,(C₁-C₆) alkenyl, (C₁-C₆) alkoxy, (C₁-C₆) alkynyl, (C₆-C₂₀) aryl,(C₆-C₂₀) substituted aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆)alkaryl, and (C₅-C₇) heteroaryl.

In one embodiment R₁, R₂, and R₃ each independently is selected from thegroup consisting of hydrogen, (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, and (C₁-C₆) alkoxy.

In another embodiment the 16-HETE analog has the following structure:

In a preferred embodiment X is NH and R₂ and/or R₃ is hydrogen. Inanother preferred embodiment R₃ is a C₃ alkyl. Preferably, the 16-HETEanalog has the following structure:

In a preferred embodiment X is O and R₂ and/or R₃ is hydrogen. Inanother preferred embodiment R₃ is a C₃ alkyl. In another preferredembodiment the 16-HETE analog has the following structure:

Several diseases or conditions are characterized by excessiveinflammation associated with neutrophil adhesion and neutrophilaggregation. The present invention provides compositions for inhibitingneutrophil adhesion and neutrophil aggregation as well as for treatingsubjects having diseases or conditions characterized by excessiveinflammation associated with neutrophil adhesion and neutrophilaggregation. In some embodiments the composition is a pharmaceuticalcomposition of a 16-HETE agonist. In these embodiments the 16-HETEagonist is present in a therapeutically effective amount for treating anadverse medical condition mediated by neutrophil adhesion and/orneutrophil aggregation. In an embodiment, the pharmaceutical preparationof the invention includes other therapeutic agents for treating theadverse medical condition mediated by neutrophil adhesion and/orneutrophil aggregation. For instance when the medical condition is aninflammatory disease the other therapeutic agents are agents fortreating an inflammatory disease or condition. According to particularembodiments of the invention the therapeutic agent for treating theinflammatory disease or condition is selected from the group consistingof antibiotics, such as tetracycline antibiotics, aminoglycosides,macrolides, lincomycins, penicillanic acid (6-APA)- derivatives having6β-acylamino groups, cephalosporanic acid (7-ACA)-derivatives having7β-acylamino groups, penicillanic acid, β-lactam antibiotics of theclavam, penem and carbapenen type, and antibiotics of the bicozamycin,novobiocin, chloramphenicol or thiamphenicol, rifampicin, fosfomycin,colistin and vancomycin and antiphlogistics, such as glucocorticoids,immunosuppressive agents, penicillamine, hydroxychloroquine, andnonsteroidal inflammation-inhibitors.

According to another embodiment, the pharmaceutical preparation includestherapeutic agents for treating an ischemic disease or condition.Preferably the therapeutic agent is selected from the group consistingof: anticoagulation agents, such as heparin, warfarin, coumadin,dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, andindandione derivatives; antiplatelet agents, such as aspirin,thienopyridine, dipyridamole and sulfinpyrazone; and thrombolyticagents, such as plasminogen, a₂-antiplasmin, streptokinase,antistreplase, tissue plasminogen activator, and urokinase.

According to one preferred embodiment of the invention, thepharmaceutical preparation includes the 16-HETE agonist of the inventiontogether with a thrombolytic agent. The pharmaceutical preparation isformulated for intravenous administration in one embodiment. In anotherembodiment the thrombolytic agent is recombinant tPA. In anotherembodiment the thrombolytic agent is a modified tPA. In one embodimentthe modified tPA is T103N, N117Q, KHRR (296-299) AAAA tPA. In anotherembodiment the modified tPA is a vampire bat tPA selected from the groupconsisting of Bat-Pa(H), Bat-Pa(I), and Bat-PA(L).

The invention also provides a composition of 16-HETE analog for use as amedicament.

The invention also provides a composition of 16-HETE agonist for use inthe manufacture of a medicament for the treatment of adverse medicalconditions mediated by neutrophil adhesion and/or neutrophilaggregation. In another embodiment the invention provides a compositionof 16-HETE agonist for use in the manufacture of a medicament for thetreatment of an inflammatory disease or condition. In another embodimentthe invention provides a composition of 16-HETE agonist for use in themanufacture of a medicament for the treatment of an ischemic disease orcondition, including but not limited to, acute stroke.

The invention also encompasses methods of treatment. According toanother aspect of the invention the 16-HETE agonist may be administeredto a subject in conjunction with other drugs for treating an adversemedical condition mediated by neutrophil adhesion and/or neutrophilaggregation. In one embodiment the condition mediated by neutrophiladhesion and/or neutrophil aggregation is an inflammatory disease orcondition. According to particular embodiments, the inflammation ischaracteristic of or results from meningitis, cerebral edema, arthritis,nephritis, adult respiratory distress syndrome, pancreatitis, myositis,neuritis, connective tissue disease, phlebitis, arteritis, vasculitis,allergy, anaphylaxis, gout, ulcerative colitis, and/or ehrlichiosis. Inone embodiment the method also includes the step of administering to thesubject a therapeutic agent other than and in addition to 16-HETEagonist for treating the inflammatory condition. According to anotherembodiment, the condition mediated by neutrophil adhesion and/orneutrophil aggregation is an ischemic disease or condition. Preferablythe ischemic condition is selected from the group consisting of a strokeand a myocardial infarction. In one embodiment the 16-HETE agonist isadministered to a subject having an ischemic disease or condition, inconjunction with a thrombolytic agent.

The 16-HETE agonist may be administered by any known method of drugdelivery. Preferably the 16-HETE agonist is administered orally orintravenously.

One aspect of the invention is directed to a method for inhibitingneutrophil adhesion and neutrophil aggregation. The method involvescontacting neutrophils with a 16-HETE agonist in situ in an amounteffective to inhibit neutrophil adhesion and neutrophil aggregation.This aspect of the invention may be applied in vitro or in vivo toinhibit neutrophil adhesion and neutrophil aggregation at a desiredtime.

In one aspect, the invention is a method for treating thromboembolicstroke. The method involves administering to a subject experiencing anacute thromboembolic stroke 16-HETE agonist in combination with athrombolytic agent in an amount effective to reduce brain injury whichwould otherwise occur as a result of the stroke. In one embodiment, the16-HETE agonist is 16(R)-HETE agonist and the thrombolytic agent is tPA.In another embodiment the 16-HETE agonist and thrombolytic agent areadministered to the subject within a first 2-6 hours after the subjectexperienced the thromboembolic stroke. In one embodiment the 16-HETEagonist is administered in an amount between 0.5 and 20 mg/kg perminute. In a preferred embodiment the 16-HETE agonist is 16(R)-HETEagonist and is administered in an amount of 1.0 mg/kg per minute. In oneembodiment the thrombolytic agent is administered in an amount between0.05 mg/kg and 1.5 mg/kg. In a preferred embodiment the thrombolyticagent is tPA and is administered in an amount of 0.9 mg/kg. In oneembodiment the thrombolytic agent is recombinant tPA. In anotherembodiment the thrombolytic agent is a modified tPA. In one embodimentthe modified tPA is T103N, N117Q, KHRR (296-299) AAAA tPA. In anotherembodiment the modified tPA. is a vampire bat tPA selected from thegroup consisting of Bat-PA(H), Bat-Pa(I), and Bat-PA(L). In yet anotherembodiment the modified tPA is C84S tPA.

According to another aspect of the invention a method of inhibitingleukotriene production in a neutrophil is provided. Leukotriene is anarachidonic acid metabolite that is a potent neutrophil chemoattractantand pro-aggregant. It was discovered according to the invention that16-HETE and agonists thereof inhibit leukotriene production inneutrophils. The method includes the step of administering to aneutrophil, 16-HETE or an agonist thereof in an amount effective toinhibit leukotriene production.

The invention in another aspect is a method of inhibiting leukotrieneproduction in a subject having a condition mediated by leukotrieneactivity. The method involves the step of administering to said subjecthaving a condition mediated by leukotriene activity a 16-HETE or anagonist thereof in an amount effective to inhibit leukotrieneproduction. In a preferred embodiment the leukotriene is leukotriene B₄.In another embodiment the leukotriene production is inhibited inneutrophils.

In one embodiment the condition mediated by leukotriene activity isselected from the group consisting of arthritis, rheumatoid arthritis,osteoarthritis, allergic rhinitis, psoriasis, dermatitis, ischemicinduced myocardial injury, reperfusion injury, gout, asthma, adultrespiratory distress syndrome, atherosclerosis, inflammatory disease,stroke, spinal cord injury, and traumatic brain injury. Preferably thecondition mediated by leukotriene activity is an inflammatory disease.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each method and product.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be more easily and completely understood whentaken in conjunction with the accompanying drawings, in which:

FIG. 1 is a bar graph depicting the percent inhibition of neutrophiladhesion to a gelatin matrix resulting from a 10 minute pre-incubationwith 16(R)-HETE, 16(S)-HETE, and 20-HETE on basal human neutrophils;

FIG. 2 is a bar graph depicting the percent inhibition of neutrophiladhesion to a gelatin matrix in response to a 10 minute pre-incubationwith 16(R)-HETE, 16(S)-HETE, and 20-HETE on thrombin-stimulated humanneutrophils;

FIG. 3 is a bar graph depicting brain infarct size in a rabbit model ofacute stroke, treated with either tPA alone (solid bar) or 16-HETE plustPA (cross-hatched bar);

FIG. 4 is a bar graph depicting regional cerebral blood flow (rCBF)immediately prior to (−0.5 hours) and at various time points followingclot embolization in a rabbit model of acute stroke, treated with eithertPA alone (cross-hatched bar) or tPA in combination with 16-HETE (solidbar), where the 16-HETE is administered between one and two hours afterclot embolization and the tPA is administered between three and fivehours after clot embolization;

FIG. 5 is a bar graph depicting the intracranial pressure (ICP)immediately prior to (−0.5 hours) and at various time points followingclot embolization in a rabbit model of acute stroke, treated with eithertPA alone (cross-hatched bar) or tPA in combination with 16-HETE (solidbar), where the 16-HETE is administered between one and two hours afterclot embolization and the tPA is administered between three and fivehours after clot embolization;

FIG. 6 is a bar graph depicting CD18 receptor density on the surface ofneutrophils isolated at various time points following clot embolizationfrom a rabbit model of acute stroke, treated with 16-HETE for one hour,beginning one hour after clot embolization; and

FIG. 7 is a bar graph depicting leukotriene B₄ synthesis in neutrophilstreated with fMLP (formyl-methionyl-leucyl-phenylalanine) alone or inthe presence of differing concentrations of 16-(R)HETE.

FIG. 8 is a bar graph depicting the ability of 16-HETE agonists toinhibit fMLP-induced neutrophil aggregation.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the finding that certain 16-HETE analogs functionas agonists and others function as antagonists. Applicants initialdiscovery that an arachidonic acid metabolic product is effective ininhibiting neutrophil adhesion and neutrophil aggregation is describedin co-pending U.S. patent application Ser. No. 08/652,327, filed May 22,1996 and issued as U.S. Pat. No. 5,753,702 on May 19, 1998 and PCTPatent Application No. PCT/US97/08865, and its related national StageU.S. patent application Ser. No. 09/194,166. Applicants also discoveredthat the compound 16-HETE, can be used effectively to inhibit neutrophiladhesion and neutrophil aggregation in both stimulated and unstimulatedneutrophils and that 16-HETE is useful as an in vivo therapeutic forreducing the extent of brain infarct damage that occurs during an acutestroke. The 16-HETE agonists described herein have the same biologicalactivity and function as 16-HETE.

The discoveries of the invention have revealed several novel propertiesof 16-HETE and analogs thereof which indicate that these molecules areparticularly advantageous as therapeutics. For instance, 16-HETE andagonists thereof are capable of inhibiting basal and thrombin-stimulatedneutrophil adhesion, and fMLP-included neutrophil aggregation. 16-HETEand agonists thereof also decrease the density of a neutrophil cellsurface receptor (CD18) involved in neutrophil adhesion. Even though16-HETE and agonists thereof have this dramatic effect on neutrophilsthese molecules do not affect platelet activity or function.Additionally, the agonists of 16-HETE have modifications which increasethe half-life of these molecules and therefore have sustainedtherapeutic value.

As demonstrated in the Examples below, 16-HETE also is capable ofinhibiting leukotriene production. When 16-HETE is administered toneutrophils, it causes a dramatic inhibition in the production ofleukotriene B₄, and therefore is useful for preventing neutrophilchemoattraction.

The compounds of the invention are 16-HETE analogs. 16-HETE has thefollowing structural formula:

The 16-HETE analogs of the invention have the following structuralformulas:

or

wherein R is selected from the group consisting of—C(O)—X—SO₂—R₁,—C(O)—X—CO—R₁, —C(O)—X—C(OH)₂—R₁, —C(O)—X—C(NH)₂—R₁, —C(O)—X—C(NH₂)₂—R₁,piperonyl, —CN, —OR′, —SR′, —NO₂, —NR′R′, amino acid, —C(O)R′, —C(S)R′,—C(O)OR′, —C(S)OR′, —C(O)SR, —C(S)SR′, —C(O)N(R′)₂, —C(O)C(O)R′,—C(S)C(O)R′, —C(O)C(S)R′, —C(S)C(S)R′, —C(O)C(O)OR′, —C(S)C(O)OR′,—C(O)C(S)OR′, —C(O)C(O)SR′, —C(S)C(S)OR′, —C(S)C(O)SR′, —C(O)C(S)SR′,—C(S)C(S)SR′, —C(O)C(O)N(R′)₂, —C(S)C(O)N(R′)₂, —C(O)C(S)N(R′)₂, or—C(S)C(S)N(R′)₂; wherein X is selected from the group consisting of O,N, and a bond; wherein R₁, R₂, and R₃ each independently is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy,aryl, and heteroaryl; wherein each R′ is (CH₂)_(z)—NR″R″ and wherein R″is independently selected from the group consisting of (C₁-C₆) alkyl,(C₁-C₆) alkenyl, (C₁-C₆) alkoxy, (C₁-C₆) alkynyl, (C₆-C₂₀) aryl,(C₆-C₂₀) substituted aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆)alkaryl, and (C₅-C₇) heteroaryl.

16-HETE analogs include both agonists and antagonists. As used herein a“16-HETE agonist” is a molecule encompassed by the above formulaswherein either R₁ or R₂ is a C₃ alkyl and the other is a hydrogen andwhich maintains the biological activity of 16-HETE. The modificationsmade to the terminal carboxyl group of 16-HETE result in a moleculehaving an equivalent or better stability than 16-HETE. In general the16-HETE agonists are more stable and have a longer half life than native16-HETE. An effective amount of a 16-HETE agonist for inhibitingneutrophil adhesion/aggregation can easily be assessed by any methodknown in the art. For example, any of the assays described in theExamples section below which examine the adhesive/aggregatory propertiesof 16-HETE on neutrophils may be utilized to assess biological activityand effective amounts. These assays include but are not limited toneutrophil adhesion, neutrophil aggregation, CD-18 expression, and LTB₄production. The 16-HETE agonists have the following general structure:

As used herein a “16-HETE antagonist” is a molecule encompassed by theabove formulas wherein when either R₁ or R₂ is a C₃ alkyl the other isnot a hydrogen. The modification at the OH group is sufficient to alterthe biological activity of the molecules from an agonist to anantagonist. The 16-HETE antagonists inhibit 16-HETE activity andtherefore prevent the 16-HETE induced inhibition of neutrophilaggregation and adhesion. An effective amount of a 16-HETE antagonistfor preventing the inhibition of neutrophil adhesion/aggregation can bedetermined in the same assays described above in relation to 16-HETEagonists except that the antagonist may be added in conjunction with16-HETE to determine if it inhibits or prevents 16-HETE activity. The16-HETE antagonists have the following general structure:

wherein R₄ and R₅ each independently is selected from the groupconsisting of hydrogen, C₁₋₂ alkyl, C₄₋₆ alkyl, alkenyl, alkynyl,alkoxy, aryl, and heteroaryl and wherein when either R₁ or R₂ is a C₃alkyl the other is not a hydrogen.

The present invention thus involves methods and products for inhibitingneutrophil adhesion and neutrophil aggregation. One method of theinvention involves administering 16-HETE agonist to neutrophils in situin an amount effective to inhibit neutrophil aggregation and neutrophiladhesion. By definition, the word “in-situ” encompasses and includes theterms “in-vivo”, “ex-vivo” and “in-vitro.”

The invention includes compositions as well as methods for treating asubject to inhibit neutrophil adhesion or neutrophil aggregation byadministering a 16-HETE agonist to a subject having an adverse medicalcondition mediated by neutrophil adhesion and/or neutrophil aggregation.In one embodiment a subject having an adverse medical condition mediatedby neutrophil adhesion and/or neutrophil aggregation is one who has aninflammatory disease or is at risk of developing an inflammatorydisease. In another embodiment a subject having an adverse medicalcondition mediated by neutrophil adhesion and/or neutrophil aggregationis one who has an ischemic disease.

A “subject” as used herein includes humans, non-human primates, dogs,cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

Both inflammatory diseases and ischemic diseases are characterized byinflammation associated with neutrophil adhesion and neutrophilaggregation due to excessive neutrophil stimulation. While not intendingto be bound by any particular theory, it is believed that excessivestimulation of neutrophils causes the neutrophils to migrate to the siteof injury, where they release toxic factors and damage surroundingtissue. When the inflammatory disease is an acute stroke a tissue whichis often damaged by neutrophil stimulation is the brain. As the activeneutrophils accumulate in the brain an infarct develops.

An “inflammatory disease or condition” as used herein refers to anycondition characterized by local inflammation at a site of injury orinfection and includes autoimmune diseases, certain forms of infectiousinflammatory states, undesirable neutrophil activity characteristic oforgan transplants or other implants and virtually any other conditioncharacterized by unwanted neutrophil activation. These conditionsinclude but are not limited to meningitis, cerebral edema, arthritis,nephritis, adult respiratory distress syndrome, pancreatitis, myositis,neuritis, connective tissue diseases, phlebitis, arteritis, vasculitis,allergy, anaphylaxis, ehrlichiosis, gout, organ transplants and/orulcerative colitis.

An “ischemic disease or condition” as used herein refers to a conditioncharacterized by local inflammation resulting from an interruption inthe blood supply to a tissue due to a blockage or hemorrhage of theblood vessel responsible for supplying blood to the tissue such as isseen for myocardial or cerebral infarction. A cerebral ischemic attackor cerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption in the blood supply tothe brain may result from a variety of causes, including an intrinsicblockage or occlusion of the blood vessel itself, a remotely originatedsource of occlusion, decreased perfusion pressure or increased bloodviscosity resulting in inadequate cerebral blood flow, or a rupturedblood vessel in the subarachnoid space or intracerebral tissue.

The methods of the invention are particularly preferred for treatingcerebral ischemia. Cerebral ischemia may result in either transient orpermanent deficits and the seriousness of the neurological damage in apatient who has experienced cerebral ischemia depends on the intensityand duration of the ischemic event. A transient ischemic attack is onein which the blood flow to the brain is interrupted only briefly andcauses temporary neurological deficits, which often are clear in lessthan 24 hours. Symptoms of TIA include numbness or weakness of face orlimbs, loss of the ability to speak clearly and/or to understand thespeech of others, a loss of vision or dimness of vision, and a feelingof dizziness. Permanent cerebral ischemic attacks, also called stroke,are caused by a longer interruption in blood flow to the brain resultingfrom either a thromboembolism or hemorrhage. A stroke causes a loss ofneurons typically resulting in a neurologic deficit that may improve butthat does not entirely resolve. Thromboembolic stroke is due to theocclusion of an extracranial or intracranial blood vessel by a thrombusor embolus. Because it is often difficult to discern whether a stroke iscaused by a thrombosis or an embolism, the term “thromboembolism” isused to cover strokes caused by either of these mechanisms. The termthromboembolism will be used throughout this patent application todescribe thrombotic and embolic strokes. Hemorrhagic stroke is caused bythe rupture of a blood vessel in a subarachnoid space or intracerebraltissue.

A preferred method of the invention involves the in vivo treatment ofthromboembolic stroke by administering to a subject experiencing anacute thromboembolic stroke a 16-HETE agonist in an amount effective toreduce brain injury which would otherwise occur as a result of thestroke. The 16-HETE agonist may be administered to the subject incombination with other therapeutics for treating acute stroke.Preferably the 16-HETE agonist is administered in combination with athrombolytic agent such as tPA.

The methods of the invention require the administration of the 16-HETEagonist in effective amounts. An effective amount of a 16-HETE agonistis one which inhibits to any degree neutrophil adhesion and neutrophilaggregation. An effective amount may be determined using the assaydescribed in Example 3 infra for neutrophil adhesion. When the 16-HETEagonist is administered in combination with a thrombolytic agent such astPA for the treatment of thromboembolic stroke an effective amount isone sufficient to reduce in vivo brain injury resulting from the stroke.A reduction of brain injury is any prevention of injury to the brainwhich otherwise would have occurred in a subject experiencing athromboembolic stroke absent the treatment of the invention. Severalphysiological parameters may be used to assess reduction of braininjury, including smaller infarct size, improved regional cerebral bloodflow, and decreased intracranial pressure, for example, as compared topretreatment patient parameters, untreated stroke patients or strokepatients treated with thrombolytic agents alone.

Effective amounts will depend, of course, on the severity of thecondition; individual patient parameters including age, physicalcondition, size and weight; concurrent treatment; frequency oftreatment; and the mode of administration. These factors are well knownto those of ordinary skill in the art and can be addressed with no morethan routine experimentation. It is preferred generally that a maximumdose be used, that is, the highest safe dose according to sound medicaljudgment, particularly if acute thromboembolic stroke is the dominantclinical manifestation.

Both the dosage and time of administration of the 16-HETE agonist andthe thrombolytic agent vary depending on a number of conditions. Acutestroke is an urgent medical condition with a small therapeutic window oftime (possibly as brief as 6 hours) in which treatment is beneficial.Clot lysing drugs are believed most useful if administered during thiswindow of time in order to at least partially restore cerebral bloodflow within the compromised region and to sustain neuronal viability.Therefore, when the thromboembolic stroke is an acute stroke it isimportant that the thrombolytic agent be administered during thecritical window of time. The dosage and time of administration of thethrombolytic agent also can be based on the target blood plasma level ofthe thrombolytic agent. Target plasma levels for humans of thethrombolytic agents are well known in the art. Studies involving thesecompounds at various dosages have been described and include Sherman D.G. et al, Chest, v. 102, p. 529S-537S; Albers, G. W., Amer. J. Card., v.75, p.34B-38B; and Saltiel and Ward, Drugs, v. 34, p. 222-262 (1987) allof which are hereby incorporated by reference. The time of theadministration of 16-HETE agonist will also vary depending on patientparameters. Generally it is most effective to begin treatment as soon aspossible after the start of the stroke.

It is expected that intravenous doses of the 16-HETE agonist in therange of 0.1 to 20 mg/kg/minute, in one or several administrations, willyield the desired results. A preferred daily dosage of 16(R)-HETEagonist is 1.0 mg/kg/minute. It is expected that intravenous doses oftPA in the range of 0.05 to 1.5 mg/kg, in one or severaladministrations, will yield the desired results. A preferred dosage oftPA is 0.9-1.1 mg/kg. In the event that the response in a subject isinsufficient at such doses, even higher doses (or effective higher dosesby a different, more localized delivery route) may be employed to theextent that patient tolerance permits. Multiple doses per day arecontemplated to achieve appropriate systemic levels of compounds.

The present invention also includes a pharmaceutical composition havinga therapeutically effective amount of 16-HETE analog included in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” as used herein means one or more compatible solid orliquid filler, dilutants or encapsulating substances which are suitablefor administration to a human or other animal. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing commingled with 16-HETE analog and with each other, in a mannersuch that there is no interaction which would substantially impair thedesired pharmaceutical efficacy.

The pharmaceutical preparation of the invention includes a 16-HETEanalog and a pharmaceutically acceptable carrier. In one embodiment, the16-HETE analog is a synthetic preparation of 16-HETE analog. As usedherein “a synthetic preparation of 16-HETE analog” includes apreparation of 16-HETE analog that is chemically derived. The chemicallyderived 16-HETE analog may be made by any chemical procedure known inthe art. An example of a procedure used to synthesize 16-HETE analog isprovided in Example 7. The compounds useful in the practice of theinvention can be prepared in accordance with the reaction described inExample 7 below or through modifications thereof, that will be readilyapparent to those skilled in the art. A suitable protocol can beselected with due consideration of the particular R, R₁, or R₂substituent, commercial availability of some starting materials, and thelike.

According to yet another embodiment, the 16-HETE analog is selected fromthe group consisting of 16(R)-HETE analog and 16(S)-HETE analog. As usedherein “16(R)-HETE analog” is an analog of the R-stereoisomer of 16-hydroxyeicosatetraenoic acid. As used herein “16(S)-HETE analog” is ananalog of the S-stereoisomer of 16-hydroxyeicosatetraenoic acid.

The pharmaceutical preparation of a 16-HETE agonist may be used alone orin combination with a therapeutic agent for treating an inflammatorydisease or condition. Known therapeutics for treating an inflammatorydisease or condition are described in medical textbooks such asHarrisons, Principles of Internal Medicine (McGraw Hill, Inc., NewYork). The particular therapeutic used depends on the nature of thedisease or condition being treated.

Therapeutics useful in the treatment of inflammatory diseases orconditions involving infectious agents include various antipathogenagents, i.e., antibiotics, antivirals, antifungals and antiparasitics.The type and concentration of therapeutic depends inter alia on theinfectious agent causing the inflammatory disease or condition. Forexample, chloramphenicol is therapeutically useful for the treatment ofmeningitis due to Strepotococcus pneumoniae, Haemophilus influenzae, andNeisseria meningitides but not in the treatment of meningitis due to E.coli or Klebsiella pneumoniae. Cefotaxime, ceftizoxime, ceftriaxone,ceftazidime, and moxalactam are useful in treating all forms ofmeningitis. Penicillin may also be used to treat S. pneumoniae and N.meningitides.

In general, therapeutics from the group comprising antibiotics include,for example, tetracycline antibiotics, such as chlortetracycline,oxytetracycline, tetracycline, demethylchlortetracycline, metacyclinc,doxycycline, minocycline and rolitetracycline; aminoglysodes, such askanamycin, amikacin, gentamicin C_(1a), C₂, C_(2b) or C₁, sisomicin,netilmicin, spectinomycin, streptomycin, tobramycin, neomycin B,dibekacin and kanendomycin; macrolides, such as maridomycin anderythromycin; lincomycins, such as clindamycine and lincomycin;penicillanic acid (6-APA)- and cephalosporanic acid (7-ACA)-derivativeshaving (6β- or 7β-acylamino groups, respectively, which are present infermentatively, semi-synthetically or totally synthetically obtainable6β-acylaminopenicillanic acid or 7β-acylaminocephalosporanic acidderivatives and/or 7β-acylaminocephalosporanic acid derivatives that aremodified in the 3-position, such as penicillanic acid derivatives thathave become known under the names penicillin G or V, such asphenethicillin, propicillin, nafcillin, oxycillin, cloxacillin,dicloxacillin, flucloxacillin, cyclacillin, epicillin, mecillinam,methicillin, azlocillin, sulbenicillin, ticarcillin, mezlocillin,piperacillin, carindacillin, azidocillin or ciclacillin, andcephalosporin derivatives that have become known under the namescefaclor, cefuroxime, cefazlur, cephacetrile, cefazolin, cephalexin,cefadroxil, cephaloglycin, cefoxitin, cephaloridine, cefsulodin,cefotiam, ceftazidine, cefonicid, cefotaxime, cefinenoxime, ceftizoxime,cephalothin, cephradine, cefamandol, cephanone, cephapirin, cefroxadin,cefatrizine, cefazedone, ceftrixon and ceforanid; and other β-lactamantibiotics of the clavam, penem and carbapenen type, such asmoxalactam, clavulanic acid, nocardicine A, sulbactam, aztreonam andthienamycin; and antibiotics of the bicozamycin, novobiocin,chloramphenicol or thiamphenicol, rifampicin, fosfomycin, colistin andvancomycin.

Anti-virals include Zidovudine (AZT-Retrovir), Zalcitabine (Hivid-ddC),Dicanosine (Videx-ddI), Protease inhibitors of retroviruses, integraseinhibitors of retroviruses and others well known to those skilled in theart.

Other therapeutics useful in the treatment of inflammatory diseases orconditions include, but are not limited to, anti-inflammatory agents, orantiphlogistics. Antiphlogistics are, for example, glucocorticoids, suchas, cortisone, hydrocortisone, prednisone, prednisolone, fluorcortolone,triamcinolone, methylprednisolone, prednylidene, paramethasone,dexamethasone, betamethasone, beclomethasone, fluprednylidene,desoxymethasone, fluocinolone, flumethasone, diflucortolone,clocortolone, clobetasol and fluocortin butyl ester; immunosuppressiveagents; penicillamine; hydroxychloroquine; and nonsteroidalinflammation-inhibitors (NSAID) which encompass anti-inflammatory,analgesic, and antipyretic drugs such as salicyclic acid, difunisal andfrom the group comprising substituted phenylacetic acid salts or2phenylpropionic acid salts, such as alclofenac, ibufenac, ibuprofen,clindanac, fenclorac, ketoprofen, fenoprofen, indoprofen, fenclofenac,diclofenac, flurbiprofen, pirprofen, naproxen, benoxaprofen, carprofenand cicloprofen; oxicam derivatives, such as piroxicam; anthranilic acidderivatives, such as mefenamic acid, flufenamic acid, tolfenamic acidand meclofenamic acid; anilino-substituted nicotinic acid derivatives,such as the fenamates miflumic acid, clonixin and flunixin;heteroarylacetic acids wherein heteroaryl is a 2-indol-3-yl orpyrrol-2-yl group, such as indomethacin, oxmetacin, intrazol,acemetazin, cinmetacin, zomepirac, tolmetin, colpirac and tiaprofenicacid; idenylacetic acid of the sulindac type; analgesically activeheteroaryloxyacetic acids, such as benzadac; phenylbutazone; etodolac;and nabumetone.

Other therapeutics useful in the treatment of inflammatory diseases orconditions include antioxidants. Antioxidants may be natural orsynthetic. Antioxidants are, for example, superoxide dismutase (SUD),21aminosteroids/aminochromans, vitamin C or E, etc. Many otherantioxidants are well known to those of skill in the art.

The pharmaceutical preparation of the 16-HETE agonist also may be usedalone or in combination with a therapeutic agent for treating anischemic disease or condition. Therapeutics for treating ischemicdiseases or conditions are described in medical textbooks such asHarrisons, Principles of Internal Medicine (McGraw Hill, Inc., New York). The particular therapeutic used depends on the nature of the diseaseor condition. Examples of therapeutics useful in the treatment ofischemic diseases or conditions include anticoagulation agents,antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Antiplatelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics and also antitlirombin agents such as, but not limited to,hirudin.

Preferably the 16-HETE agonist is administered in conjunction with athrombolytic agent when treating an ischemic disease. Thrombolyticagents lyse clots which cause the thromboembolic stroke. Thrombolyticagents have been used in the treatment of acute venous thromboembolismand pulmonary emboli and are well known in the art (e.g. see Hennevekenset al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, etal, J Am Coll Cardiol; v.25 (7 suppl), p. 10S-17S(1995)). Thrombolyticagents include, but are not limited to, plasminogen, a₂-antiplasmin,streptokinase, antistreplase, tissue plasminogen activator (tPA), andurokinase.

In a preferred embodiment of the invention tPA is the thrombolyticagent. The mature tPA polypeptide has 527 amino acids, at least 17 (Asn)of which have been shown to be linked with carbohydrate structures.Spellman et al., have identified several of these carbohydrates,including a high-mannose structure on amino acid 117, and di-tri-andtetra-antennary N-acetyllactosamine-type structures on amino acids 184and 448 [J. Biol. Chem. 264 (24) 14100-14111 (1989)].

“tPA” as used herein includes native tPA and recombinant tPA, as well asmodified forms of tPA that retain the enzymatic or fibrinolyticactivities of native tPA. The enzymatic activity of tPA can be measuredby assessing the ability of the molecule to convert plasminogen toplasmin. The fibrinolytic activity of tPA may be determined by any invitro clot lysis activity known in the art, such as the purified clotlysis assay described by Carlson, et. al., Anal. Biochem. 168, 428-435(1988) and its modified form described by Bennett, W. F. Et al., 1991,Supra, the entire contents of which are hereby incorporated byreference.

Recombinant tPA has been described extensively in the prior art. Severalforms of recombinant tPA are commercially available such as ACTIVASE®.

Modified forms of tPA (“modified tPA”) have been characterized and areknown to those skilled in the art. Modified tPA includes, but is notlimited to, variants having deleted or substituted amino acids ordomains, variants conjugated to other molecules, and variants havingmodified glycosylation. Several preferred modified tPAs have beendescribed in PCT Publication No. W093/24635; EP 352,119; EP382174; andSuzuki et al., J. Cardiovasc. Pharmacal. 22, 834-840 (1993). Each ofthese references is hereby incorporated by reference.

Briefly, PCT Publication No. W093/24635 discloses tPA variants having anextra glycosylation site at any of the amino acid positions 103-105 andthe native glycosylation site removed at position 117 of the nativehuman tPA. The amino acid number refers to the amino acid in thatposition of the mature, wild-type tPA polypeptide as disclosed in U.S.Pat. No. 4,766,075. These variants have extended circulatory half livesand exhibit substantially the same or improved fibrin binding affinityand fibrinolytic potency as compared to wild-type human tPA. Thedisclosed variants may also include at least one amino acid substitutedin the 296-299 position with alanine and/or a substitution of the aminoacids at positions 274-277 of wild type tPA (phenylalanine, arginine,isoleucine, lysine) with leucine, histidine, serine, and threonine,respectively. One particularly effective type of variant disclosed inthe reference is a triple mutant variant of wild type tPA. The firstmutation in a triple mutant is the addition of one glycosylation site atleast one of the amino acid positions 103-105 by e.g., substituting thenative amino acid sequence 103 with an asparagine as part of anAsn-X-Ser or Asn-X-Thr tripeptidyl sequence, wherein X is any amino acidexcept proline. The second mutation involves the removal of aglycosylation site at native amino acid site 117 (Asn) and replacing itwith another amino acid, preferably glutamine. The third mutation is thereplacement of native amino acids 296-302 with other amino acids. Themost effective of the triple mutant variants is the specific molecule,T103N, N117Q, KHRR (296-299) AAAA tPA (TNK tPA).

EP 352,119 discloses Vampire Bat tPA's (Bat-Pa (H), (I), and (L)).Vampire bat-Pa's are variants of native tPA having a variety of sequencemodifications. Although the Bat-Pa variants are structurally distinctfrom tPA because they lack the Kringle 2 domain and plasmin-sensitiveprocessing site, these variants are functionally similar to native tPA.They are however, more potent than native tPA.

Suzuki et al., J. Cardiovasc. Pharmacal. 22, 834-840 (1993) disclose tPAvariants in which a cysteine at position 84 of the growth factor domainof native tPA is replaced by scrine (C84S tPA). Although this variantretains the functional activity of native tPA, it has been shown to havea longer in vivo half life than native tPA.

The 16-HETE agonist may be administered alone or may be delivered in amixture with other drugs, such as those disclosed above, for treatingthe inflammatory or ischemic disease or condition. In some embodiments,a common administration vehicle (e.g., pill, tablet, implant, injectablesolution, etc.) would contain both the 16-HETE agonist useful in thisinvention and the therapeutic for treating the inflammatory or ischemicdisease or condition. Thus, the present invention also providespharmaceutical compositions, for medical use, which comprise the 16-HETEagonist of the invention together with one or more pharmaceuticallyacceptable carriers thereof and optionally other therapeuticingredients.

The invention also includes compositions and methods for treating asubject to prevent 16-HETE inhibition of neutrophil adhesion orneutrophil aggregation and is therefore useful for immunostimulation bypromoting neutrophil activity at sites of infection.

In addition to the therapeutic uses of the 16-HETE analogs, thesecompounds are also useful for a variety of in vitro purposes. Forexample, these compounds are useful in competition assays as well asintermediates or starting material for the synthesis of other compounds.

The invention also encompasses methods of inhibiting leukotrieneproduction in a neutrophil by contacting the neutrophil with 16-HETE ora 16-HETE agonist. Leukotrienes are arachidonic acid metabolites havingpotent biological activity. The leukotrienes have been implicated in avariety of disease states including, for example, inflammation.Leukotriene B₄ is a potent chemotactic factor for inflammatory cells andhas been found in the synovial fluids of rheumatoid arthritis patientsand in psoriatic lesions. Leukotriene C₄ and D₄ have been demonstratedto be involved in constriction of human airway smooth muscle cells. Themethod of inhibiting leukotriene production in a neutrophil or in asubject having a condition mediated by leukotriene activity involves thestep of administering a 16-HETE or an agonist thereof in an amounteffective to inhibit leukotriene production. The leukotriene may be anytype of leukotriene, but preferably is leukotriene B₄. A conditionmediated by leukotriene activity is one selected from the groupconsisting of allergic rhinitis, adult respiratory distress syndrome,inflammatory bowel disease, ischemic induced myocardial injury,reperfusion injury, gout, asthma, psoriasis, stroke, spinal cord injuryinflammation and traumatic brain injury. Preferably that disorder is aninflammatory disease. The treatment of diseases by promoting leukotrieneproduction is described in U.S. Pat. No. 5,466,669 which is herebyincorporated by reference.

Methods for assessing leukotriene B₄ inhibition in neutrophils includethe assay set forth below in Example 6. Many in vitro assays ofleukotriene activity inhibition are also known in the art. These includefor example, the rat and dog ex vivo leukotriene B4 inhibition assaysset forth in U.S. Pat. No. 5,612,377 issued to Crooks et al., which ishereby incorporated by reference.

When administered, the formulations of the invention are applied inpharmaceutically acceptable amounts and in pharmaceutically acceptablecompositions. Such preparations may routinely contain salts, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic ingredients. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic,p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic,succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal(0.004-0.02% W/V).

Generally, daily oral doses of active compounds will be from about 0.01milligrams/kg per day to 1000 milligrams/kg per day. It is expected thatoral doses in the range of 50 to 500 milligrams/kg, in one or severaladministrations per day, will yield the desired results. Dosage may beadjusted appropriately to achieve desired drug levels, local orsystemic, depending upon the mode of administration. In the event thatthe response in a subject is insufficient at such doses, even higherdoses (or effective higher doses by a different, more localized deliveryroute) may be employed to the extent that patient tolerance permits.Multiple doses per day are contemplated to achieve appropriate systemiclevels of compounds.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular combination ofdrugs selected, the severity of the condition mediated by neutrophiladhesion and/or neutrophil aggregation being treated, the condition ofthe patient, and the dosage required for therapeutic efficacy. Themethods of this invention, generally speaking, may be practiced usingany mode of administration that is medically acceptable, meaning anymode that produces effective levels of the active compounds withoutcausing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, topical, nasal, direct injection,transdermal, sublingual or other parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion. Intravenous and intramuscular routes are not particularlysuited for long term therapy and prophylaxis. They could, however, bepreferred in emergency situations such as a stroke or myocardialinfarction. Direct injection could also be preferred for local deliveryto the site of injury. Oral administration may be preferred forprophylactic treatment because of the convenience to the patient as wellas the dosing schedule.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the 16-HETE analog intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the 16-HETE analog into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the 16-HETE analog, which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. can be found inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the 16-HETE analog. Othercompositions include suspensions in aqueous liquors or non-aqueousliquids such as a syrup, an elixir, or an emulsion.

The 16-HETE agonist and thrombolytic agent or other therapeutic usefulin the treatment of inflammatory or ischemic diseases may beadministered by the same method, e.g. intravenous, oral, etc. or may beadministered separately by different modes, e.g. 16-HETE agonistadministered orally, thrombolytic agent administered intravenously, etc.In one embodiment of the invention the 16-HETE agonist and thethrombolytic agent or other therapeutic are co-administeredintravenously. In another embodiment the 16-HETE agonist and thethrombolytic agent or other therapeutic are administered separately.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the 16-HETE analog of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di-and tri-glycerides; liposomes; phospholipids; hydrogel release systems;silastic systems; peptide based systems; wax coatings, compressedtablets using conventional binders and excipients, partially fusedimplants and the like. Specific examples include, but are not limitedto: (a) erosional systems in which the polysaccharide is contained in aform within a matrix, found in U.S. Pat. Nos. 4,452,775, 4,675,189, and5,736,152, and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions mediated by neutrophiladhesion and/or neutrophil aggregation. “Long-term” release, as usedherein, means that the implant is constructed and arranged to delivertherapeutic levels of the active ingredient for at least 7 days, andpreferably 30-60 days. The implant may be positioned at the site ofinjury. Long-term sustained release implants are well known to those ofordinary skill in the art and include some of the release systemsdescribed above.

EXAMPLES Example 1 Identification and Quantitative Analysis ofNeutrophil Arachidonic Acid Metabolites

Materials

HETE standards (16(R)-, 16(S)-, 17-, 18-, 19- and 20-HETE) and19-HETE-d₃ (99+atom % deuterium) were synthesized as described in Falckel al., J. Biol. Chem. 265, 10244-10249 (1990). The standardlipoxygenase-derived HETEs (5-, 8-, 11-, 12-, and 15-HETE) were obtainedfrom Biomol. Thrombin and FMLP were purchased from Parke-Davis (MorrisPlains, N.J.). All solvents used were HPLC grade (Burdick and Jackson,Muskegon, Mich.) and other reagents were of the highest gradecommercially available. BSTFA was purchased from Aldrich and [1-¹⁴C]arachidonic acid (55 mCi/mmol) was purchased from du Pont Corp.(Wilmington, Del.). All other reagents were purchased from SigmaChemical Co., St. Louis, Mo. unless otherwise noted.

Methods

Preparation of Human Polymorphonuclear Leukocytes

Venous blood (60-70 ml) was collected from four healthy, normalvolunteers into syringes containing heparin (1 U/ml). Polymorphonuclearleukocytes (PMNs) were isolated by a histopaque density gradienttechnique as previously described in Liu et al., Adhesion, Its role inInflammatory Disease, Freeman and Co., New York, 1992, pp 189-192.Briefly, blood aliquots (6 ml) were layered over two layers (3 ml) ofhistopaque 1077 and 1119 in a conical centrifuge tube. The sample wasthen subjected to centrifugation at 100×g for 30 minutes at roomtemperature and the top layer, containing the neutrophils, was carefullycollected. Hypotonic lysis was used to separate out the red blood cells.The neutrophils were then isolated from the sample by centrifugation(2000×g) and suspended in Haiks balanced salt solution (HBSS) containing1 mM Ca²⁺ and 0.8 mM Mg²⁺ for studies involving arachidonic acidmetabolism, and in M199 (with 1% BSA) for neutrophil adhesion assays.Total cells were counted using a hemocytometer and leukocytes werecounted after staining with Wright-Giemsa stain. For each experiment,the cell preparations used contain≧97% neutrophils, having≧95% viabilityas determined by a trypan blue exclusion assay.

Arachidonic Acid Metabolism

Neutrophils were suspended in HBSS (10⁷ cells per ml) and allowed tostand for 10 minutes. Radioactively labeled arachidonic acid (1 to 50μM) was produced with either a ¹⁴C label containing 0.5 to 1 μCi[1-¹⁴C]arachidonic acid/1 to 50 μM unlabeled arachidonic acid] or adeuterium label (1 unit arachidonic acid-d₈/2 units unlabeledarachidonic acid). The experimental samples were preincubatcd withinhibitors [SKF 525A (100 μM) or BW755C (94 μM) or indomethacin (10 μM)]for 10 minutes prior to the addition of arachidonic acid. At the end ofthe incubation, the labeled arachidonic acid, was added to theneutrophil suspension and allowed to incubate for 10 minutes at 37° C. Acontrol sample was not subjected to preincubation with inhibitors.

After ten minutes in arachidonic acid the reaction was terminated by theaddition of cold methanol (four volumes). The neutrophil methanolmixture was then subjected to acidification at pH 3.5 to 4 and themetabolite fraction was extracted with ethyl acetate. The extracts werewashed with water and dried over anhydrous sodium sulfate. Themetabolites were further purified by filtration to remove the sodiumsulfate and evaporation to remove the ethyl acetate. The resultantresidue was dissolved in methanol and injected on a reverse phase HPLCcolumn (ODS silica, 250×4.6 mm, Beckman). Eluate was collected at 1ml/min. The solvent gradient was started at acetonitrile/water/aceticacid (62.5:37.5:0.1) and increased to 100% acetonitrile in 20 minutes.The effluent was passed through a UV detector(HP1050/ChemStation—Hewlett-Packard, Palo Alto, Calif.) and aradioactivity detector (Radiomatic, Meridian, Conn.) with a splitter(ratio 1:10) and collected in 1 ml fractions.

Gas Chromatography/Mass Spectrometry

The fractions containing radioactive metabolites were evaporated undervacuum and derivatized as follows before being subjected to massspectrometric analyses: Methyl esters were prepared with diazomethanesolution in ether (100 μl, 3 min); Pentaflurobenzyl esters (PFB) wereprepared with pentafluorobenzyl bromide and N,N-diisopropylethylamine asdescribed Balazy, J. Biol. Chem., 266, 23561-23567 (1991); andTrimethylsilyl (TMS) ethers of hydroxyl groups were prepared withN,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA). The derivativeswere then dissolved in isooctane and analyzed by GC/MS as describebelow. In some experiments, aliquots of isooctane solution were mixedwith catalytic amounts of 5% rhodium on alumina (Aldrich, Milwaukee,Wis.) in a micro-vial and bubbled with hydrogen gas at 0° C. for 5 minBalazy and Murphy Anal. Chem. 58, 1098-1100 (1986). The catalyst wasprecipitated by centrifugation and the reduced derivative was analyzedby GC/MS.

Aliquots (1 μl) were injected into a 15 m fused silica GC column (DB-1,0.25 mm i.d., 0.25 μm film thickness, J and W Scientific, Folsom,Calif.) and eluted with a flow of helium (44 cm/s) and with atemperature program from 170° C. to 300° C. (rate 15° C./min). The massspectrometer was operated in chemical ionization mode with negative iondetection (electron capture) using methane as a reagent gas (2.6 torrsource pressure) or in electron ionization mode at 70 eV. The relativeretention time of each derivative was expressed as carbon numberequivalent calculated from retention times obtained for a series ofmethyl or PFB esters of saturated fatty acids (C₁₄-C₂₄) as described(Balazy, supra).

Quantitative Analysis of 20-HETE and 16-HETE in Neutrophil Phospholipids

Suspensions of human neutrophils (0.5 to 0.9×10⁸ cells per ml) fromthree healthy donors not receiving medication were extracted using theBligh and Dyer procedure as described Zhu et al., Hypertension 25,854-859 (1995). The neutrophil total lipids containing 0.1 to 0.3 μmolof phosphorus, were treated with 200 μl of 0.1 N potassiumhydroxide/ethanol (1:1) or water/ethanol (in control experiments) for 1hr at 50° C., and extracted with ethyl acetate after acidification (pH3-4). Prior to extraction, internal standard, 19-HETE-d₃ (2 ng), wasadded. The extracts were dried and purified by RP-HPLC. Followingderivatization with PFB bromide and BSTFA, the samples were analyzed byGC/MS as described (Zhu, supra). The amount of endogenous HETE wascalculated from the standard curve prepared with 2 ng of 19-HETE-d₃ and19-HETE (0.1 to 1 ng). The amount of phosphorus in the samples analyzedby GC/MS was determined spectrophotometrically (Zhu, supra).

Stereochemical Analysis of 16-HETE

The radiolabeled material in fraction 10, 16(R)-HETE, 16(S)-HETE and20-HETE were separately esterified with pentafluorobenzyl bromide andthe hydroxyl group was further esterified with a-naphthoyl chloride asdescribed Laethem et al., J. Biol. Chem. 268, 12912-12918 (1993). Thederivatives were purified by reverse-phase HPLC and analyzed onchiral-phase column (Pirkle type 1-A, 250×4.6 mm, Regis, Morton Grove,Ill.) and cluted with hexane containing 0.1% of isopropanol at 1 ml/min.The effluent was analyzed by a UV detector and collected in 0.2 mlfractions. The amount of radioactivity in these fractions was measuredusing scintillation counter.

Results

Identification of Neutrophil Arachidonic Acid Metabolites

Previously, Bednar et al. demonstrated that incubation of arachidonicacid with intact canine and human neutrophils produce polar metaboliteswhose formation can be inhibited by SKF525A or carbon monoxide (Bednaret al., Biochem. Biophys. Res. Commun. 123, 581-588 (1984); Bednar etal., Biochem. Pharmacol. 36, 1741-1747 (1987); and Kraemer, supra).These studies were later extended in human neutrophils. The latterstudies revealed that intact neutrophils contain cytochrome P450monooxygenase system and produce a cytochrome P450-dependentarachidonate metabolite which was found to inhibit neutrophilaggregation (Kraemer, supra, Bednar et al., Biochem. Pharmacol. 36.1741, 1987). The work of Hatzelmann and Ullrich (Hatzelmann, supra)showed that intact human neutrophils metabolize arachidonic acid intotwo major compounds: 20-HETE, an omega-hydroxylase product, and 15-HETE,a lipoxygenase product. Surprisingly the inventors of the presentinvention found that neither 20-HETE nor 15-HETE inhibited neutrophilaggregation.

In order to identify the neutrophil arachidonic acid metabolitesresponsible for inhibiting neutrophil aggregation, human neutrophilswere incubated with radiolabeled arachidonic acid. Incubations ofarachidonic acid with human neutrophils resulted in formation of fourradiolabeled products (metabolites A, B, C, and D). The major peak (D)contained unmodified arachidonic acid. Peaks B and C combined contained5 to 8% of the total radioactivity injected and displayed absorbance at234 nm, which is typical of conjugated double bonds. The material inpeak A contained 10 to 15% of the radioactivity and it did not absorb UVlight above 205 nm. Preincubation with inhibitors, established that theformation of product A by human neutrophils can be inhibited withSKF525A but not with BW755C suggesting that product A originated frommetabolism mediated by cytochrome P450 monooxygenase, and not bylipoxygenase or cyclooxygenase.

Metabolite A was subjected to GC/MS analysis in order to furtherelucidate the components of metabolite A. The GC/MS analysis ofmetabolite A as a PFB, TMS derivative, with monitoring of ion m/z 391,which corresponded to a PFB ester, TMS ether derivative of a HETEmolecule, produced an ion chromatogram which indicated the presence oftwo components at retention times of 7.33 min (component A1) and 8.01min (component A2), having carbon number equivalents 20.9 and 22.4,respectively. Although variable amounts of metabolite A was isolatedfrom neutrophils of each of the individual donors, the GC/MS analysesconsistently yielded components A1 and A2 with a relative abundance ofapproximately 1 to 4. When deuterium-labeled arachidonic acid wasincluded in the incubations, additional clromatographic peaks wereobserved (corresponding to ion m/z 399) which had retention timessimilar but slightly shorter than the non-labeled analogs, possibly as aresult of the isotopic effect of the deuterium substitution.

The gas chromatographic mobility was compared with that of HETEstandards. This comparison revealed that the component A2 coeluted with20-HETE while the component A1, coeluted with 16-HETE. The PFB, TMSderivative of a standard sample of lipoxygenase HETEs (containing equalamount of 5-, 8-, 12-, and 15-HETE) eluted as a single peak at 7.45 minand did not coelute with either component of metabolite A or either ofthe 20- or 16-HETEs.

The electron capture spectra obtained for both the A1 and A2 componentscontained prominent ions at m/z 391 (M-181, loss of pentafluorobenzyl)and at m/z 301 (M- 181-90, loss of pentafluorobenzyl andtrimethylsilanol, SiMe₃OH) and were consistent with molecular weight of320 amu for the metabolite having one hydroxyl group in the arachidonicacid structure. Inclusion of arachidonic acid-d₈ in the incubationsproduced a spectrum containing additional isotopic ion at m/z 399. Whensynthetic 16-HETE was mixed with metabolite A, only the intensity ofcomponent A1 increased and the added compound could not be separatedfrom biologically derived A1, strongly indicating that Product A1 was16-HETE. Catalytic reduction of PFB, TMS derivative resulted in a shiftby 8 mass units producing a spectrum with ion m/z 399, typicallyobserved for a HETE molecule with four double bonds reduced.

To further characterize these two components, the metabolite A wasmethylated, silylated with BSTFA and catalytically reduced withhydrogen. The electron ionization mass spectrometry of component A1(carbon number equivalent 22.3) produced ions at m/z 399 (M-CH₃), m/z357 and m/z 159 which originated from α-cleavage at C16, m/z 235 (m/z357-32-90, loss of CH₃OH and SiMe₃OH). Fragment m/z 357 displayed acomplex isotopic cluster of ions resulting from scrambling of deuteriumduring catalytic hydrogenation of isotopically-labeled molecule.Fragment ion m/z 328 originated from rearrangement of trimethylsilylgroup to carboxylic group similar as observed for saturated hydroxyfatty acids. Eglinlon et al., Org. Mass. Spectrom. 1, 593-611 (1968).The spectrum of synthetic, reduced 16-HETE, revealed the presence ofthese ions at retention time equivalent to carbon number 22.3 and wassimilar to the spectrum published by Falck et al., supra.

The stereochemistry of the hydroxyl at C16 in component A1 wasestablished by chiral phase HPLC. The mixing of metabolite A withracemic synthetic 16-HETE followed by derivatization into 16-naphthoyl,PFB ester resulted in separation of this derivative into enantiomers andrevealed that the radioactivity from biologically derived compoundcoeluted with the R isomer. The 20-naphthoyl, PFB derivative of 20-HETEwas completely separated from the 16-HETE derivative during RP-HPLCpurification prior to chiral analysis. These data were consistent withthe structure of metabolite A1 as16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid formed by the action ofcytochrome P450 on arachidonic acid assuming that the configuration ofdouble bonds was unchanged from that of arachidonic acid. The electronionization of the reduced molecule of component A2 as methyl ester TMSderivative (carbon number equivalent 23.5) revealed ions at m/z 414(M⁺), m/z 399 (M-CH₃), m/z 367 (M-CH₃—CH₃OH), m/z 324 (M-SiMe₃OH), m/z292( M-32-90, loss of CH₃OH and SiMe₃OH), m/z 146 (SiMe₃O(CH₂)₃),CH₃)m/z 103 (Me₃Si—O═CH₂). The isotopic cluster of ions m/z 399 and 367resulted from the scrambling of deuterium-labeled molecule duringcatalytic reduction of double bonds. This spectrum was consistent withreduced methyl ester, TMS ether derivative of a mixture of 20-HETE and20-HETE-d₈.

Electron ionization of reduced metabolite B as methyl ester, TMS etherproduced a spectrum (carbon number equivalent 21.8) which containedprominent ions at m/z 414 (M⁺), 399 (M-15), 343 (α-cleavage at C15) anda base peak at m/z 173 (SiMe₃OCH(CH₂)₄CH₃). Ion m/z 343 displayeddistinct isotopic cluster as expected for a reduced deuterium-labeledmolecule. It was concluded that the metabolite B was 15-HETE.

The material in metabolite C contained at least two components absorbingUV light at 234 nm. The latter eluting component coeluted with standard5-HETE. The material in peak C produced a complex mass spectrumindicative of 5-HETE and another unidentified product. Longerincubations of arachidonic acid with human neutrophils also resulted information of a polar compound identified as 20-carboxy arachidonic acidwhich originated from oxidative metabolism of 20-HETE.

Therefore, the mass spectrometric analyses of arachidonic acidmetabolites produced by intact human neutrophils obtained from threehuman donors, revealed that a single monitoring of ion m/z 391,corresponding to a PFB, TMS derivative of a HETE molecule, consistentlyshowed a minor, less polar component in addition to 20-HETE. Using massspectrometric analysis and chiral analysis, it was revealed that theminor component was 16(R)-HETE, a product of cytochrome P450 andarachidonic acid which has not been previously observed in human PMN.Although, two recent studies provided mass spectrometriccharacterization (Hatzelmann, supra) and quantitation of 20-HETE inhuman PMNs (Hill, supra), 16-HETE was not observed. The lack ofdetection of 16-HETE is most likely due to the similar chromatographicmobility of 20-HETE and 16-HETE during RP-HPLC which results in theinability to observe separate peaks for these two HETEs using a lowresolution of the radioactivity detector. It was also found in thepresent study that gas chromatographic conditions were capable of fullyseparating derivatives of unreduced 20-HETE from four subterminal HETEs.These chromatographic conditions were different than those described forisolation of 20-HETE from human neutrophils (Hatzelmann, supra, Hill,supra).

Example 2 16-HETE and 20-HETE are Released from Neutrophil Phospholipids

Methods

Quantitative analysis of 20-HETE and 16-HETE in neutrolphilphospholipidswas performed as described above in Example 1.

Results

Release of Endogenous 16-HETE and 20-HETE from Neutrophil Phospholipids

When total lipid extracts from intact neutrophils were purified by HPLCand analyzed by electron capture GC/MS, no HETE molecules were detectedby this GC/MS assay and therefore must be less than 10 pg per 10⁸ cells.However, when neutrophil lipids were hydrolyzed with 0.1 N potassiumhydroxide, 20-HETE and 16-HETE were detected at a concentration of341±69 pg per 10⁸ cells and 108±26 pg per 10⁸ cells respectively (Table1).

TABLE 1 Amounts of 16-HETE and 20-HETE released from intact humanneutrophil phospholipids following alkaline hydrolysis 16-HETE 20-HETE(pg/10⁸ cells) (pg/10⁸ cells) control n.d. n.d. Hydrolysis 108 ± 26 341± 69 Average ± SEM of single measurements of neutrophil samples obtainedfrom three donors. HETEs were measured by gas chromatography/massspectrometry with 19-HETE-d₃ as an internal standard as described in themethods above.

Example 3 16-HETE Inhibits Neutrophil Adhesion

Methods

Synthesis of 16-HETE

Nucleophilic S_(N)2 addition of butyl magnesium bromide in Et₂O to theacetalic center of homochiral 1,3-dioxolan-4-one, prepared from3-methyl-2-butenal and (R)-mandelic acid, followed by esterificationwith diazomethane readily afforded substituted O-allyl mandelate.Diisopropyl acetal was then obtained via low temperature ozonolysis,with Me₂S workup and incubation of the resultant crude aldchyde withtrilisopropyl orthoformate in the presence of catalytic pyridiniump-toluenesulfonate (PPTS). Mild oxidative decarboxylation via an insitu-generated dioxetanone led to the corresponding benzoate. Minoramounts of free alcohol released during this process were benzolylatedand the combined degradation product was hydrolyzed, usingtrifluoracetic acid to furnish the aldehyde. Wittig condensation with13-carbomethoxytrideca-3(Z), 6(Z),9(Z)-trien-1-ylidene-triphenylphophorane and methanolysis of thebenzoate produced methyl 16(S-OH-AA(8), [α]_(D)=−5.4 (c 0.7, acetone).Mitsunobu inversion (PhCO₂H,Ph₃P, DEAD) and benozate removal (NaOMe,MeOH, 24° C., 2h) yielded the 16(R)-isomer.

Neutrophil Adhesion

Isolated neutrophils were resuspended in M199 containing 1% BSA with afinal concentration of 3×10⁶ cells per ml. Cells suspensions (1 ml) wereincubated with or without a test compound, 16(S)-HETE, 16(R)-HETE, or20-HETE (concentrations 0.01 to 10 μM) for 10 min and then placed intogelatin-coated wells and further incubated for 20 min at 37° C. Thewells were washed three times with 0.5 ml of solution M199 to removenon-adherent cells. The adherent cells were then removed by addition of0.4 ml of 0.25% trypsin-EDTA in 0.1 M PBS to the gelatin-coated wellsand incubation for 10 min at 37° C. The incubates were transferred andthe wells were washed with 0.1 ml of M199 solution. The amount of cellsin combined solutions was counted. In some experiments, the adhesion ofneutrophils was stimulated by addition of 1 U/ml of thrombin toneutrophil suspension immediately prior to the transfer of cells to thewells. Experiments were performed in duplicates for each concentrationof HETE. The inhibition of adhesion was expressed as percent of cellsremaining in suspension (non-adherent) relative to control. The range ofadherent PMNs in control incubations was 6 to 23%. This was increasedtwo-fold in the presence of thrombin.

Neutrophil Aggregation and Chemiluminescence

Stimulated neutrophil aggregation and oxygen free radical release wereexamined in whole human blood using a whole-bloodaggregometer-chemiluminometer (Chronolog, Haverton, Pa.) as described inBednar et al. J. Thromb. Thrombol. 1, 179-185 (1995). Briefly, bloodsamples containing heparin (1 U/ml) as the anticoagulant were diluted inHBSS in proportion 1 to 9. Diluted blood aliquots (1 ml) were stirred(400 rpm) in a prewarmed (37° C.) siliconized cuvette containingcytochalasin b (5 μg/ml) and luminol (in 8 μl DMSO, 500 μM finalconcentration) and the test HETE (concentration 0.01 to 10 μM) for 10min. Neutrophils were stimulated with a submaximal dose of fMLP (5×10⁻⁷Mfinal concentration). The aggregation and chemiluminescence weresimultaneously measured as changes of impedance and luminescence,respectively. The fMLP-induced chemiluminescence was calculated as thepeak height of the reaction.

Results

Neutrophil-inhibitory Activity of 16-HETE

In order to determine the biological effects of 16-HETE on humanneutrophils, neutrophil adhesion was analyzed after preincubation of16-HETE and 20-HETE. At concentrations≦20 μM, 16-HETE (R or S) and20-HETE had no detectable agonist activity on human neutrophils.However, both stereoisomers of 16-HETE inhibited basal (FIG. 1) andthrombin-stimulated (FIG. 2) neutrophil adhesion to gelatin matrix.Treatment of neutrophils with 16(R)-HETE or 16 (S)-HETE atconcentrations from 10⁻⁸ to 10⁻⁵M inhibited neutrophil adhesion in adose-dependent maimer. The 16(R)-HETE at a concentration of 1 μMsignificantly (p<0.05) inhibited basal neutrophil adhesion by 73±11% andthrombin-stimulated adhesion (1 U/ml, 3×10⁶ cells/ml) by 50±4%. Incontrast to the 16(R)-HETE, the 16(S)-HETE was less potent in inhibitingunstimulated (39±8%) and thrombin-stimulated (40±14% inhibition)neutrophil adhesion at a concentration of 1 μM, although it exhibitedthe same biological activity as 16(R)-HETE. Preincubation of neutrophilswith 10 μM of 16(R)-HETE completely inhibited neutrophil adhesion togelatin. 20-HETE at concentrations ≦10 μM did not inhibit neutrophiladhesion under either basal or stimulated conditions.

The 16(R)-HETE also inhibited fMLP-induced neutrophil aggregation (Table2) with IC₅₀ of 1.2 μM. The 16(S)-HETE and 20-HETE were not active atconcentrations of ≦10 μM. Finally, the fMLP-stimulated release of oxygenfree radicals was not inhibited by any of the three HETEs tested atconcentrations≦10 μM (Table 2).

The effect of preincubation with 16(R)-HETE, 16(S)-HETE and 20-HETE onneutrophil aggregation and oxygen free radicals (OFR) release stimulatedby fMLP (5 × 10⁻⁷M, final concentration) in diluted human bloodAggregation OFR Release 16(R)-HETE  42.0 ± 11.0* 8.9 ± 5.1  16(S)-HETE7.0 ± 3.5 5.6 ± 16.7 20-HETE 8.1 ± 2.6 3.9 ± 13.4 Values representpercent of inhibition from HETE untreated cells (N = 3 to 4 in eachgroup). 1 mM concentrations of HETE was used for each analysis. OFRrelease was measured by chemiluminescence as described above. *(p <0.05)

The biological effects of 16-HETE on human neutrophils have not beenpreviously reported. 16(R)-HETE displayed a potent inhibitory activitytowards unstimulated and stimulated neutrophils. The biological effectobserved with 16(R)-HETE was more potent than the effect observed withthe other stereoisomer, 16(S)-HETE. 16(R)-HETE inhibited neutrophiladhesion, basal and thrombin-stimulated, and fMLP-induced neutrophilaggregation.

It was observed that in order to inhibit the adhesion of neutrophils toa gelatin matrix, a 10 min preincubation with 16(R)-HETE was required.This could reflect the time necessary for the 16-HETE to achievemembrane concentration sufficient for inhibition of adhesion. While notintending to be bound by any particular theory, the data presented abovesuggest that 16-HETE inhibits neutrophil activation indirectly, throughinduction of changes in phospholipid membrane, possibly viaincorporation into sn-2 position of lyso-phospholipids. The resultspresented here clearly demonstrate that neutrophil lipids containdetectable quantities of 16-HETE and 20-HETE which must originate fromendogenous arachidonic acid and exist as preformed cellular componentsin a form sensitive to alkaline hydrolysis.

In summary we have shown that 16(R)-HETE is formed in human neutrophilsfrom arachidonic acid, it is an endogenous compound, and it has potentanti-adhesion and antiaggregatory properties. These observationsindicate that 16-HETE is an important effector in regulating thephysiological response to human neutrophil activation.

Example 4 Intravenous Administration of 16-HETE in Combination with tPAis More Effective than tPA in Reducing Brain Injury in a Rabbit Model ofThromboembolic Stroke

Methods

New Zealand rabbits of either sex, weighing 3-4 kilograms were used forthe study. Prior to the start of the experiment, the rabbits weredivided into three groups, those that were administered both 16-HETE ata rate of 1 mg/kg per minute for one hour and tPA (Genentech, Inc., SanFrancisco, Calif.) at a concentration of 6.3 mg/kg, tPA alone, orvehicle.

On the day of treatment the animals were fasted for 12 hours with freeaccess to water and then anesthetized with an intramuscular solution ofketamine (50 mg/kg), acepromazine (20 mg) and xylazine (5 mg/kg). Thissolution was subsequently used to maintain a surgical depth ofanesthesia as determined by responses to various physiologic andautonomic stimuli, including mean arterial pressure and response to pawpinch.

The animals were prepared for surgery by introducing femoral venous andarterial catheters (PE50 and 90, respectively; Clay Adams, Parsippany,N.J.). The catheters were placed for subsequent blood sampling,including blood gas and arterial pressure, drug infusions, and fluidreplacement.

All animals were tracheotomized and mechanically ventilated in order tomaintain arterial blood gases within a physiologic range. Arterial bloodgas measurements (pH, pCO₂, pO₂) were determined, using a Corning 168blood gas monitor. Arterial blood gases were maintained withinphysiologic range throughout the protocol with hematocrit and arterialpressure maintained near initial base line values.

A midline scalp incision was made to expose the calvarium. Bilateralcraniectomies were performed in which a temperature sensor was placed inorder to measure brain temperature and a 30-gauge platinum-iridiumelectrodes were inserted 2 mm within the cortical mantle to monitorregional cerebral blood flow via the hydrogen clearance technique. Allthe instrumentation was carefully fixed in place with fast-settingepoxy. Both core and brain temperatures are maintained within 1° C. ofbase line values through the use of heating blankets and heating lamps.

Once the surgical procedures had been prepared, the instrumentation wasallowed to equilibrate for 30 minutes, prior to embolization.

A clot embolus was prepared 4 hours prior to the embolization procedureby mixing 1 cc of donor rabbit blood with 20 μM tin granules (50 mgtin/ml whole blood) and placed in PE 90 tubing pretreated with thrombin(Park-Davis, Morris Plains, N.J.).

One hour after embolization 16-HETE was continuously infused into theanimals at a rate of 1 mg/kg per minute for 1 hour. The thrombolytic tPAwas infused between 3 and 5 hours following embolization at a dose of6.3 mg/kg. The animals received either 16-HETE, tPA, 16-HETE plus tPA,or vehicle. Measurements were taken for a total of 7 hours followingclot embolization.

Following determination of all base line values, the right commoncarotid bifurcation is exposed, the internal carotid artery (ICA)isolated, a micro-arteriotomy performed and the 15-mm clot embolizedintracranially via the ICA. ICA flow is then reestablished via amicro-arteriorrhaphy. Post embolization rCBF is then determined and theexperiment continued if rCBF is reduced to 15 cc/100 gm/minute in atleast one of the PT-IR electrodes within the embolized hemisphere. Thevalue of rCBF reduction is based on historical studies demonstrating theintensity of ischemia necessary to produce irreversible brain injury, ifno therapeutic intervention is instituted. A submental vertex X-ray,using a Phillips dental X-ray machine, verified intracranial placementof the tin-tagged clot. Submental vertex X-rays were repeated every 30minutes until termination of the experiment. Care was taken to maintainthe same position of the X-ray tube relative to the skull. Clot lysiswas defined as complete dissolution of the embolus.

At the completion of the study the animals were euthanized, acalveroectomy was performed, and the brain was harvested. The brain wascarefully inspected and X-rayed for the presence and position of aresidual clot. The brain was then subjected to 2-mm coronal sectioningand incubated in isosmolar TTC at 37° C. for 30 minutes to delineate theregion of infarct. Infarct size is determined according to themodification described by Lin et al., Stroke, 24:117-121, 1993. Eachbrain section was also examined for gross hemorrhage.

At the time of embolization, the common, external, and internal carotidarteries were all transiently excluded from the circulation and the clotembolus injected into the anterior circulation of the brain via theinternal carotid artery. Immediately following clot embolization,regional cerebral blood flow was measured by the hydrogen clearancetechnique (Young W., Stroke, 11:552-564, 1980) within the embolizedhemisphere in order to insure a reduction in regional cerebral bloodflow to ≦15 cc/100 g/min. Additionally, a submental vertex X-rayconfirmed intracranial clot placement. All animals met both criteria.Internal carotid artery patency was restored using 10-0 interruptednylon sutures (Sharpoint, Reading, Pa.).

Statistical analysis of all hematological variables, ex-vivo neutrophilfunction, cerebral blood flow, and infarct size was performed usingrepeated measures analysis of variance (ANOVA) to detect significantdifferences between groups and among time points.

Results

Hematocrit, arterial blood gases, and mean arterial pressure weremonitored and controlled prior to and following clot embolization. Theseparameters, as well as core and brain temperature, were well maintainedthroughout the experimental protocol and generally showed no statisticalsignificance in experimental vs. control groups.

Brain infarct size was measured following completion of the surgicalprocedure. Infarct size in both the tPA alone and vehicle groups wassignificantly larger than the 16-HETE and tPA group (FIG. 3). Braininfarct size was found to be related to the final regional cerebralblood flow (rCBF) value (FIG. 4). Animals in the 16-HETE and tPA groupexhibited both the smallest infarct size and the greatest improvement inrCBF as a percentage of the base line value. 16-HETE also reducedintracranial pressure (ICP).(FIG. 5)

Example 5 16-HETE Transiently Decreases CD18 Receptor Density inNeutrophils

Methods

Animals were studied as described above in Example 4, except that theanimals were administered 16-HETE alone at a dose of 1 mg/kg/min for onehour. Neutrophils were isolated at the following time points: 0.5 hourprior to embolization and 0.5, 1.5, 2.5 and 4 hours followingembolization.

CD18 receptor density was determined by the following binding assay.Whole blood samples were collected at the time points outlined for theneutrophil activation studies. An aliquot of 100 μl from each sample waspromptly treated with a saturating dose of primary monoclonal anti-CD11/18 antibody (WMRD, Inc., Pullman, Wash.) at 1 μg/ 10⁶ cells.Following a thirty minute incubation at 4° C., each preparation waswashed with 2 ml of cold Dulbecco's Phosphate Buffered Solution (GibcoBRL, Grand Island, N.Y.). It was then centrifuged for seven minutes at7° C., 1500 rpm. After discarding the supernatants, a secondarymonoclonal anti-IgG fluorescein conjugated antibody (Dako Corporation,Carpinteria, Calif.) was added to the samples. This was followed by asecond 30 minute incubation at 4° C. The preparations were rewashed andcentrifuged in the same manner as previously stated. Again discardingthe supernatant, 250 μl of Optilyse C (Immunotech, Inc., Westbrook, Ma.)was added to lyse the erythrocytes and fix the remaining cells andantibody reactions. The cells next incubate at room-temperature fortwenty minutes, and were rewashed and centrifuged for the final time.After removal of the supernatant, the cells were resuspended in 1 ml of2% formalin (Baxter Healthcare Corporation, McGaw park, Ill.) diluted inDPBS. Throughout the study, three additional samples were prepared usinga primary murine IgGI antibody (Caltag Laboratories, Burlingame,Calif.). Its use in place of the primary monoclonal anti-CD 11/18antibody, served as an isotypic control (‘IC’) to estimate nonspecificbinding of the primary antibody. The IC samples were processed in thesame manner as those above. All final samples were wrapped in foil andstored at 4° C. in preparation for flow cytometric analysis thefollowing morning. Total sample analysis on the Coulter Epics Eliterequires thirty minutes. Neutrophils were identified by theirdistinctive forward versus side scattered signal. The CD 11/18positivity was determined based upon these gated events. A standardcurve was generated using Quantum 26 Fluorescent Beads (Flow CytometryStandards Corporation, San Juan, PR). Actual MESF values were determinedusing the QuickCal 2.0 software package (Flow Cytometry StandardsCorporation).

Results

The ability of a neutrophil to adhere to a vessel wall is mediated byCD1 8 receptors on the neutrophil surface. Following clot embolizationin an animal model of acute stroke, CD18 receptor density issignificantly increased, causing an increase in the ability of aneutrophil to adhere to a vessel wall. When 16-HETE is administeredafter clot embolization, CD18 receptor density was found to decreasesignificantly (FIG. 6). The decrease in CD18 receptor density occurredimmediately after the administration of 16-HETE and was transient. After16-HETE administration was stopped, CD18 receptor density returned topre-treatment levels.

The fact that the change in CD18 density in response to 16-HETEtreatment is so rapid and is transient holds important clinicalsignificance for the treatment of acute stroke. Neutrophils serve manyimportant physiological functions in regulating inflammation. Althoughit is important to reduce neutrophil aggregation for the treatment ofacute stroke, it is important to be able to restore neutrophil activityquickly if necessary. The regulation of neutrophil function is adelicate balance which could easily upset the physiological mechanismsinvolved in many processes. This balance is not easy to a achieve usingdrugs with prolonged activity. Therefore, the discovery that 16-HETEinduces a rapid physiological response which is halted immediately afterremoval of the drug has important clinical significance.

Example 6 16-HETE Transiently Decreases CD18 Receptor Density inNeutrophils

Methods

Neutrophil LTB₄ Production

16(R)-HETE (0.01-1.0 μM) was preincubated for 10 minutes with theneutrophil suspension (3×10⁶ /ml) prior to the addition offormyl-methionyl-leucyl-phenylalanine (fMLP). Samples were thenincubated for an additional 10 minutes with 5×10⁻⁷M fMLP. Allincubations took place at 37° C. Plasma samples for the measurement ofLTB₄ were collected by centrifugation of the whole blood samples at 1200rpm for 15 minutes. Samples were stored at 70° C. until analyzed. Theplasma was then acidified to pH3 with 1M Hcl. The plasma was then placedon C2 reverse phase columns (Amprep for Amersham Corp., ArlingtonHeights, Ill.) to extract LTB₄. LTB₄ was then measured using the BiotrakLTB₄ enzyme immunoassay system (Amersham Corp., Arlington Heights,Ill.). All standards and samples were performed in duplicate. Followingincubation, all samples were read at 450 nm and the LTB₄ concentrationin each sample was determined following the generation of a standardcurve. LTB₄ concentration was expressed as picograms LTB₄ per 1000neutrophils.

Results

The addition of fMLP (5×10⁻⁷M) to isolated PMN suspensions resulted inan approximate doubling of the LTB₄ synthesis with levels of 92.8±27.1pg/1000 neutrophils noted in the stimulated group when compared to alevel of 49.9±24.2 pg/1000 PMNs in the unstimulated control group (FIG.7). Preincubation with 16(R)-HETE resulted in a dose related inhibitionof LTB₄ synthesis that was nearly complete at a concentration of 1 μM(17.0±3.2 pg/1000 neutrophils; p<0.05; n=4).

16-HETE potently suppressed LTB₄ production in neutrophils. LTB₄ is apotent neutrophil chemoattractant and pro-aggregant (Ford-Hutchinson, A.W.; Bray, M. A.; Doig, M. V.; Shipley, M. E.; and Smith, M. J. H.Leukotriene B, a potent chemokinetic and aggregating substance releasedfrom polymorphonuclear leukocytes. Nature 286:264-265 (1980). Theinhibition of LTB₄ by 16-HETE contributes to its suppression ofneutrophil activation. Thus the present studies support a regulatoryrole for 16-HETE in neutrophil function. It should be noted that themajor cytochrome P450 product in neutrophils, 20-HETE, had no activitywhen examined in assays of neutrophil aggregation, adhesion or luminolchemiluminescence at concentrations up to 1 μM.

Example 7 Synthesis of 16-HETE Analogs

Methods

An equimolar mixture of 16-hydroxyeicosatetracnoic acid (16 HETE) andN-hydroxysuccinimide in anhydrous tetrahydrofuran (THF) was cooled to 0°C. To this was added 1 equivalent of dicyclohexylcarbodiimide (DCC) withstirring. After 12 h at room temperature, the solvent was removed invacuo and the residue was purified by SiO₂ chromatography to afford thecorresponding 16-HETE N-hydroxysuccimide ester in 75% yield as acolorless oil.

The above active ester (1 equivalent), methanesulfonamide (10equivalents), and 4-(dimethylamino)pyridine (1 equivalent) were heatedat 90 ° C. for 1.5 h in a minimum of dry hexamethylphosphoramide (HMPA).The cooled reaction mixture was added to water and extracted 3 timeswith EtOAc. The combined organic extracts were evaporated in vacuo andthe residue purified by SiO₂ chromatography to affordN-methylsulfonyl-16-hydroxyeicosa-5,8,11,14-(Z)-tetraenamide as acolorless oil in 54% yield.

Example 8

Methods

To a compound (YKR-II-230-30) (2-55 mmol; 500 mg) in solvent THF: HMPAmixture (4:1 total 40 ml) at −40° C. ⁷BuLi (3.06 m mole; 2.5 solution inHexane) was added dropwise and stirring was continued for 1 hr at sametemperature. Then bromide (JY-I-27-20) (3.06 mmol; 1.572 gms) in THF (5ml) was added at −40° C. and reaction mixture slowly allowed to roomtemperature and stirring was continued for overnight. Then reactionmixture cooled to 0° C., quenched with sat, aq, NhuCI solution,extracted with ether, washed with water, brine, dried over Na₂SO₄,concentrated and purified on silicagel column chromatography using EtoAcand N-Hexane or elements to get pure YKR-II-258-34 in 76% yield (1-22gms) as colorless liquid.

Example 9

Methods

To a solution of compound (YKR-II-258-34) (0.64 mmole, 400 mg) in THF(15) ml at 0° C. TBAF (Tetrabutyl Ammonium Fluoride) (3.183 mmol; 1.0 msolution in THF; 3.183 ml) was added. Then reaction mixture was allowedto room temperature and stirrings was continued for overnight. Thensolvent was removed under vacuum, diluted with CH₂J₂, washed with water,brine, dried over Na₂SO₄ and concentrated. The residue was purified onsilicagel column chromatography using EtoAc and N-Hexane or elements toget pure YKR-II-258-34 in 87% yield (216 mg) as colorless syrup.

Example 10

Methods

To a solution of compound (YKR-II-261-26) (0.26 mmol; 100 mg) in DMF (5ml) at room temperature PDC (Pydridinium diclomate)(1.28 mmole; 4.82 mg)was added and stirring was continued for 14 hours at same temperature.Reaction mixture then diluted with ether, washed with water, brine,dried over Na₂SO₄ and concentrated. Then the residue was treated withCH₂N₂ at 0° C. in Et₂O:MeOH (4:1) (total:10 ml) mixture. Then solventwas removed under vacuum and the residue was purified on silicagelcolumn chromatography using EtoAc and N-Hexane solvents to getYKR-II-263-36 in 66% yield (70 mg).

Example 11

Methods

To a solution of compound (YKR-II-263-36) (0.099 mmol; 40 mg) inMethanol (20 ml) at 0° C. catalytic amount of PTSA (P-Towene Sulphonicacid) was added and reaction mixture allowed to room temperature.Stirring was continued for 2 hrs. Solvent was removed under vacuum,residue was dissolved in EtoAc, washed with NaHCO₃ solution, brine,dried over sat. aq. Na₂SO₄ and concentrated. The crude residue waspurified on PTLC to get the pure compound YKR-II-266-31 in 92% yield (29mg).

Example 12

Methods

To a solution of compound YKR-II-266-31 (0.016 mmole: 5.2 mg) in THF:H₂Omixture (5:1; Total 5 ml) at 0° C., 1.0 m solution of aq. LiOH (0.047mmole; 47 ml) was added and reaction mixture allowed to roomtemperature. Stirring was continued for 12 hrs; the reaction mixturecooled to 0° C., neutralized with 1.0 m aq. oxalic acid solution.Volatiles were removed, diluted with EtoAL, washed with water, brine,dried over Na₂ SO₄, concentrated and purified on PTLC to getYKR-II-285-27 in 82% (4 mg) yield.

Example 13

Methods

To a stirred solution of Nickelacetate tetrahydrate (0.04 mmoles; 9.9mg) in EtOH(20 ml)at room temperature NaBHu (0.04 mmoles; 1.3 mg) wasadded under Hydrogen atmosphere. After 30 minutes stirring Ethylenediamine (0.08 mmoles: 5.3 ml) was added, followed by addition ofcompound (YKR-II-258-34)(0.79 mmoles; 500 mg). After two hours ofstirring, reaction mixture diluted with others and filtered through bedof silicagel solvent was removed under reduced pressure. Crude mixturewas purified on silicagel column chromatography using EtoAc and N-Hexaneor element to get the pure compound YKR-II-262-27 in 93% yield as acolorless syrup.

Example 14

Methods

To a solution of compound (YKR-II-262-27) (0.08 mmoles; 50 mg) in THF(10 ml) at 0° C. TBAF (Tetrabutyl ammonium fluoride) (0.40 mmoles; 1.0 msolution in THF: 400 ml) was added. Then reaction mixture allowed toroom temperature and stirring was continued for overnight. The solventwas removed under vacuum, diluted with CH₂J₂, washed with water, brine,dried over Na2SO4, concentrated and the residue was purified on PTLC toget the pure compound YKR-II-280-26 in 85% yield (26.5 mg) as colorlesssyrup.

Example 15

Methods

To a solution of compound (YKR-II-280-26) (0.05 mmoles; 20 mg) inmethanol (5 ml) at 0° C., catalytic amount of PTSA (P-Towene SulphonicAcid) was added and reaction mixture was allowed to room temperature.Stirring was continued for two hrs. Solvent was removed, residue wasdissolved in EtoAc, washed with sat. aq. NaHCO₃ solution, brine, driedover Na₂SO₄ and concentrated. The crude residue was purified on PTLC toget YKR-II-251-25 in 92% yield (14.5 mg) as pure compound.

Example 16

Methods

To a solution of compound (YKR-II-264-26) (0.13 mmoles; 50 mg) in DMF (5ml) at room temperature PDC (Pyridinium dichromate)(0.63 mmoles; 238.7mg) was added and stirring was continued for 16 hrs at same temperature.Then reaction mixture was diluted with ether, washed with water, brine,dried over Na₂SO₄ and concentrated. The residue was treated with CH₂N₂at 0° C. in Et₂O MeOH (5:1) mixture. Solvent was removed and the residuewas purified on PTLC to get YKR-II-282-34 as pure compound in 68% yield(36 mg).

Example 17

Methods

To a solution of compound (YKR-II-282-34) (0.05 mmoles; 20 mg) inmethanol (10 ml) at 0° C. catalytic amount of PTSA was added andreaction mixture allowed to room temperature. Stirring was continued for2 hrs. Solvent was removed, residue was dissolved in EtoAc, washed withsat. aq. NaHCO₃ solution, brine, dried over Na₂SO₄, concentrated andpurified on PTLC to get the compound YKR-II-283-27 in 89% yield (14.3mg).

Example 18

Methods

To a solution of compound (YKR-II-283-27) (0.013 mmoles; 4.4 mg) in THF:H20 mixture (5:1; Total 5 ml) at 0° C., 1.0 m solution fo aq. LiOH(0.039 mmoles, 39 ml) was added and reaction mixture allowed to roomtemperature. Stirring was continued for overnight. Then reaction mixturerecooled to 0° C., neutralized with 1.0 m aq. oxalic acid solution.Volatiles were removed, diluted with EtoAc, washed with water, brine,dried over Na₂SO₄, concentrated and the residue was purified on PTLC toget the compound YKR-II-284-27 in 86% yield (3.6 mg).

Example 19

Methods

To a solution of compound (YKR-II-267-26) (0.07 mmoles; 30 mg) inmethanol (5 ml), 10% pd-c (5 mg) was added and reaction mixture wasstilTed under hydrogen atmosphere for 2 hrs. Then reaction mixturefiltered through a pad of celite, concentrated and purified on PTLC toget the compound YKR-II-271-28 in 94% yield (28.7 mg) as white solid.

Example 20

Methods

To a solution of compound YKR-II-271-28 (0.047 mmoles, 20 mg) inmethanol (5 ml) at 0° C., catalytic amount of PTSA was added. Reactionmixture then allowed to room temperatures, stirring was continued for 2hrs. Solvent was removed, diluted with EtoAc, washed with sat. aq.NaHCO₃ solution, brine, dried over Na2SO₄, concentrated and the residuewas purified on PTLC to get the compound (YKR-II-272-26) in 87% yield(14 mg) as white solid.

Example 21

Methods

To a solution of compound YKR-II-272-26 (0.014 mmoles, 4.9 mg) in THF:H₂O mixture (5: 1; Total 5 ml) at 0° C., 1.0 m solution of aq. LiOH(0.043 mmoles; 43 μl) was added and reaction mixture allowed to roomtemperature. Stirring was continued for 12 hrs. Then reaction mixturerecooled to 0° C. and neutralized with 1.0 m. aq. oxalic acid withwater, brine, dried over Na2SO4, concentrated and the residue waspurified on PTLC to get the compound YKR-II-286-27 in 85% yield (4.0 mg)as white solid.

Example 22 16-HETE Analogs Which Retain Neutrophil Inhibiting Activityof 16-HETE

Methods

The methods were performed as described above in Example 3.

Results

In order to demonstrate the biological effects of 16-HETE on humanneutrophils, neutrophil aggregation was analyzed after preincubation of16-HETE(R) and 5 separate 16-HETE analogs. At concentrations of onemicromolar, 16-HETE (R) and each analog inhibited FMLP-inducedneutrophil aggregation (Table 3 and FIG. 8). The data is based on fourindependent observations per compound tested. Although the sulfonamidederivative had slightly lower activity than 16-HETE (R), it stilldemonstrated significant therapeutic potential.

TABLE 3 The effect of preincubation with 16(R)-HETE and 16-HETE analogson neutrophil aggregation Aggregation 16(R)-HETE  42.0 ± 11.0*8,9,11,12-tetrahydro-16(R)-HETE  71.1 +/− 10.3*5,6,14,15-Dehydro-8,9,11,12-tetrahydro-16(R)-HETE 79.4 +/− 12.2C(1)-hydroxyl 16-HETE 84.6 +/− 11.45,6,8,9,11,12,14,15-octahydro-16(R)-HETE 122.8 +/− 15.4  16(R)-HETEsulfonimide  47.6 +/− 20.5* Values represent percent of inhibition fromHETE untreated cells (N = 4 in each group). 1 mM concentrations ofcompound was used for each analysis. *(p < 0.05)

The biological effects of 16-HETE on human neutrophils have not beenpreviously reported. 16(R)-HETE displayed a potent inhibitory activitytowards unstimulated and stimulated neutrophils. The biological effectobserved with 16(R)-HETE was more potent than the effect observed withthe other stereoisomer, 16(S)-HETE. 16(R)-HETE inhibited neutrophiladhesion, basal and thrombin-stimulated, and fMLP-induced neutrophilaggregation.

Example 23 16-HETE does not Affect Platelet Function

Methods

Platelet aggregation was examined in diluted whole blood using aChronology whole blood aggregometer (Havertown, Pa.) as previouslydescribed (Bednar, M. M.; Dooley, R. H.; Zamani, M.; Howard, D. B.; andGross, C. E. Neutrophil and platelet activity and quantificationfollowing delayed t-PA therapy in a rabbit model of thromboembolicstroke. J. Thromb. Thrombol. 1:179-185 (1995). Briefly, whole blood wascollected in 3.8% sodium citrate (9:1, v/v) and was subsequently diluted50:50 with normal saline. Aggregation was assessed over a 6 minuteinterval following addition of a submaximal concentration of adenosinediphosphate (ADP, 5 μM: Sigma Corporation, St. Louis, Mo.). Aggregationwas then repeated following a ten minute incubation with either 1 μM or16(R)-HETE or 16(S)-HETE and expressed as the change in impedance as apercentage of the baseline value. Adenosine triphosphate (ATP) releasewas simultaneously measured from ADP-stimulated platelets in theChronology whole blood aggregometer using the Chrono-Lume luminescentagent (firefly luciferin-luciferase). Samples were compared tochemiluminescence obtained from ATP standards following the addition ofluciferin-luciferase.

Results

No significant effect of either 16(R)-HETE or 16(S)-HETE was seen onplatelet function at a concentration of 1 μM (n=3, mean±sem). Whencompared to ATP release in control samples, 16(R)-HETE and 16(S)-HETEreleased 99.3±0.7 and 108.0±6.8% of the control sample respectively.Similarly, ADP-induced platelet aggregation following incubation with 1μM 16(R)- and 16(S)-HETE was 85.9±21.4 and 101.3±14.0% of the controlvalue, respectively.

Various metabolites of arachidonic acid demonstrates overlappingprofiles of biological activity. It has previously been demonstrated at15-HETE (Takata, S.; Matsubara, M.; Allen, P. G.; Janmey, P. A.; Serhan,C. N.; Brady, H. R. Remodeling of neutrophil phospholipids with15(S)-hydroxyeicosatetracnoic acid inhibits leukotriene B4-inducedneutrophil migration across endothelium. J. Clin. Invest. 93:499-508(1994); Petrich, K.; Ludwig, P.; Kuhn,; H.; Schewe, T. The suppressionof 5-lipoxygenation of arachidonic acid in human polymorphonuclearleukocytes by the 15-lipoxygenase product 15(S)-hydroxy-5Z, 8Z, 11Z,13E)-cicosatetraenoic acid: structure-activity relationship andmechanism of action. Biochemical Journal 314:911-6 (1996); and Huang, Z.H.; Bates, E. J.; Ferrante, J. V.; Hii, C. S. T.; Poulos, A.; Robinson,B. S.; and Ferrante, A. Inhibition of stimulus-induced endothelial cellintercellular adhesion molecule-1, E-selectin, and vascular cellularadhesion molecule-1 expression by arachidonic acid and its hydroxy andhydroperoxy derivatives. Cir. Res. 80:149-158 (1997) and prostacyclin(Darius, H.; Veit, K.; Binz, C.; Fish, A.; and Meyer, J. Diminishedinhibition of adhesion molecule expression in prostacyclin receptordesensitized human platelets. Agents & Actions 45:77-83 (1995); andBoxer, L. A.; Allen, J. M.; Schmidt, M.; Yoder, M.; and Baehner, R. L.Inhibition of polymorphonuclear leukocyte adherence by prostacyclin. J.Laboratory & Clinical Med. 95:672-678 (1980) may both downregulateadhesion receptors and suppress neutrophil function. Althoughsuppression of neutrophil function is shared by various eicosanoids,including 16-HETE in the present study, the 16-HETE biological activityappears to be relatively specific for the neutrophil as prostacyclininhibits platelet activation (Tateson, J. E.; Moncada, S.; Vane, J. R.Effects of prostacyclin (PGX) on cyclic AMP concentrations in humanplatelets. Prostaglandins 13:389-397 (1977)) whereas 15-HETE has beendemonstrated to increase platelet activation (Setty, B. N.; Werner, M.H.; Hannun, Y. A.; and Stuart, M. J. 15-Hydroxyeicosatetracnoicacid-mediated potentiation of thrombin-induced platelet functions occursvia enhanced production of phosphoinositide-derived secondmessengers—-sn-1,2-diacylglycerol and inositol-1,4,5-triphosphate. Blood80:2765-2773 (1992)). Studies in our laboratory also suggest that16-HETE does not possess significant vasoactive properties unlike thesignificant hypotension seen with prostacyclin (Armstrong, J. M.;Lattimer, N.; Moncada, S.; and Vane, J. R. Comparison of vasodepressoreffects of prostacyclin and 6-oxo-prostaglandin F1 alpha with those ofprostaglandin E2 in rats and rabbits. Brit. J. Pharm. 62:125-130 (1978))in vivo or enhanced the vascular reactivity seen with 15-HETE in vitro(Uski, T. K.; and Hogestatt, E. D. Effects of various cyclooxygenase andlipoxygenase metabolites on guinea-pig cerebral arteries. Gen. Pharm.23:109-113 (1992) and Van Diest, M. J.; Herman, A. G.; and Verbeuren, T.J. Influence of hypercholesterolemia on the reactivity of isolatedrabbit arteries to 15-lipoxygenase metabolites of arachidonic acid:comparison and platelet-derived agents and vasodilators. ProstaglandinsLeukocytes & Essential Fatty Acids 54: 135-145 (1996)). This relativespecificity for neutrophil function is advantageous in both determiningcell mechanisms as well as designing therapies for various ischemicstates.

STATISTICS

Student's t test was used to compare control versus 16-HETE-treatedsamples. Differences were considered significant at the p<0.05 level.

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

We claim:
 1. A compound of a 16-hydroxyeicosatetraenoic acid analogcomprising a compound selected from the group consisting of:


2. The compound of claim 1 wherein the 16-hydroxyeicosatetraenoic acidanalog is an agonist having the following structure:


3. The compound of claim 1 wherein the 16-hydroxyeicosatetraenoic acidanalog is an agonist having the following structure: